JP2002543093A - Compositions and methods for treating cancer by selective inhibition of VEGF - Google Patents

Abstract

(57) SUMMARY Disclosed are antibodies that specifically inhibit VEGF binding to only one of two VEGF receptors (VEGFR2). These antibodies efficiently inhibit angiogenesis and further have improved safety due to their specificity. Thus, the present invention provides new antibody-based compositions, methods and combined protocols for treating cancer and other angiogenic disorders. Advantageous immunoconjugate and prodrug compositions and methods of using these new VEGF-specific antibodies are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

This application claims priority to co-pending US Provisional Application No. 60 / 131,432, filed April 28, 1999, and disclaims the entire text and drawings of this application. No particular reference is made herein by reference. The United States Government has assigned the National Institutes of Health assign numbers 1RO1 CA74951, 5RO CA54168 and T32
In accordance with GM07062, the rights in the present invention are reserved.

1. FIELD OF THE INVENTION [0002] The present invention relates generally to the fields of antibody, angiogenesis and tumor treatment. More specifically, the present invention provides an anti-VEGF antibody that specifically inhibits VEGF, which binds to only one of the two VEGF receptors (VEGFR2). Such antibodies inhibit angiogenesis and induce tumor regression, and further have improved safety due to their specific locking properties. The antibody-based compositions and methods of the present invention also extend to the use of immunoconjugates and other therapeutic conjugates, kits and methods containing the conjugates, including prodrugs.

2. Description of the Related Art Tumor cell resistance to chemotherapeutic agents represents a significant problem in clinical oncology.
Indeed, this is one of the major causes that, despite certain advances in the field, many of the most prevalent forms of human cancer are still resistant to effective chemotherapy.

[0004] Another tumor treatment strategy is the use of "immunotoxins", where anti-tumor cell antibodies are used to deliver toxins to tumor cells. However, in common with chemotherapeutic approaches, immunotoxin treatment also suffers significant disadvantages when applied to solid tumors. For example, antigen-negative or antigen-deficient cells can persist and regrow the tumor or lead to further metastasis.

[0005] A further reason for the resistance of solid tumors to antibody-based therapies is that the tumor mass is generally impermeable to macromolecules such as antibodies and immunotoxins (Burrows et al., 1992; Dvorak et al., 1991a; Baxt.
er and Jain, 1991). Both the physical diffusion distance within the tumor and the interstitial pressure are significant limitations for this type of treatment. Thus, solid tumors, which make up more than 90% of all human cancers, have more proven resistance to antibody and immunotoxic treatments.

[0006] More recent strategies have targeted the angiogenesis of solid tumors. Targeting the blood vessels of the tumor, rather than the tumor cells themselves, is unlikely to lead to the development of resistant tumor cells and has certain advantages in that the target cells are readily acceptable. In addition, because many tumor cells rely on a single blood vessel for their oxygen and nutrients, vascular destruction leads to amplification of the antitumor effect (Bur
rows and Thorpe, 1994a; 1994b). Representative vascular targeting strategies are described in U.S. Patent Nos. 5,855,866 and 5,965,132.
, Which specifically describe the targeted delivery of anticellular drugs and toxins to markers of tumor blood vessels.

[0007] Another effective version of the vascular targeting approach is to target clotting factors to markers expressed or adsorbed in cellular blood vessels (Huang et al., 1).
997; U.S. Patent Nos. 5,877,289, 6,004,555, and 5,, (US Application No. 08 / 482,369 (Oct. 1998)
Payment of patent issuance fee on March 20) Delivering a coagulant rather than a toxin to tumor vessels has the added benefit of reduced immunogenicity and a lower risk of toxin side effects. As described in US Pat. No. 5,877,289, preferred coagulation factors for use in such tumor-specific "coagulants" include truncated forms of human coagulation-inducing protein, tissue factor (TF), blood coagulation Is the main initiator.

[0008] Although the specific delivery of toxins and coagulation factors to tumor blood vessels represents a significant advance in tumor treatment, certain peripheral tumor cells can outweigh the intratumoral destruction caused by such treatments. Accordingly, anti-angiogenic strategies are described in US Pat. No. 5,855,86.
No. 6 and 5,877,289.

[0009] Anti-angiogenic tumor treatment strategies are based on inhibiting the growth of sprouting blood vessels, generally around solid tumors. These treatments are used primarily to reduce the risk of micrometastases or to inhibit the further growth of solid tumors following more conventional interventions (eg, surgery or chemotherapy).

[0010] Angiogenesis is the development of new blood vessels from existing blood vessels and / or circulating endothelial stem cells (Asahara et al., 1997; Springer et al., 1998; Fo).
lkman and Shining, 1992). Angiogenesis plays an important role in many physiological processes, such as embryogenesis, wound healing and menstruation. Angiogenesis is also important in certain pathological phenomena. In addition to their role in solid tumor growth and metastasis, other notable conditions with angiogenic components are arthritis, psoriasis, and diabetic retinopathy (Hanahan and Folkman, 1
996; Fidler and Ellis, 1994).

Angiogenesis is regulated in normal and malignant tissues by a balance of angiogenesis stimulants and angiogenesis inhibitors produced in target and distant sites (Fidler et al., 1998; McNamara et al., 1998).
Vascular endothelial growth factor-A (also known as VEGF, vascular permeability factor, VPF)
It is the primary stimulator of angiogenesis. VEGF is a multifunctional cytokine caused by basal oxygen and tumor mutations and can be produced by various tissues (Kerbel et al., 1998; Mazure et al., 1996).

[0012] Recognition of VEGF as a primary stimulator of angiogenesis in pathological conditions has been
Various attempts have been made to block activity. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers to VEGF, and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors are all proposed for use in interfering with VEGF signaling ( Siemeister et al., 1998). Indeed, monoclonal antibodies to VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (Kim et al., 1993; Asano et al., 1998; Mesia)
no et al., 1998; Luo et al., 1998a; 1998b; Borgstrom et al., 1996; 1998).

[0013] Although the above studies highlight the importance of VEGF in solid tumor growth and its potential as a target for tumor therapy, the identification of additional agents that inhibit VEGF-induced angiogenesis has This is an advantage in expanding the number of therapeutic options. The development of therapeutic agents that more specifically inhibit VEGF receptor binding represents a significant advance, unless their antitumor effects are substantially impaired by improved specificity.

SUMMARY OF THE INVENTION The present invention overcomes certain deficiencies in the prior art by providing novel therapeutic compositions and methods for use in anti-angiogenic and anti-tumor treatments. The present invention is based on antibodies that specifically inhibit VEGF, which bind only to one of the two primary VEGF receptors (VEGFR2). Such antibodies inhibit angiogenesis and induce tumor regression as effectively as other anti-VEGF antibodies, including those already in clinical trials, and furthermore, their specific blocking properties Has improved safety. The compositions and methods of the present invention also include
It extends to the use of immunoconjugates and combinations (including prodrugs) using specific classes of provided antibodies.

[0015] A particular advantage of the present invention is that the provided antibodies do not bind to VEGFR1,
To inhibit VEGF, which binds only to GFR2. This is VEGFR2
Prior art major antibodies that inhibit VEGF binding to both and VEGFR1 (
A4.6.1). Since VEGFR1 has an important biological role independent of angiogenesis, particularly in macrophage migration and chemotaxis, and osteoclast and cartilage resorption functions, the current ability to inhibit only VEGFR2-mediated angiogenesis is Another advantage. This translates to a remarkable clinical advantage in that macrophages can further mediate the host antitumor response, and that bone metabolism is not adverse, for example, in the treatment of childhood cancer.

A further advantage is that because VEGF binding to VEGFR1 is maintained in the presence of the antibodies of the invention, they bind to VEGF bound to VEGFR1, which is up-regulated on tumor endothelium. Used to specifically deliver therapeutic agents to tumor blood vessels. Thus, in the context of an immunoconjugate, the present invention provides an agent having both anti-angiogenic and tumor destructive properties in the same molecule.

[0017] There are additional advantages in the ability of the compositions provided to neutralize VEGF survival signals mediated by VEGFR2. Thus, the naked conjugated antibodies of the present invention are synergistic with other therapeutics and / or binders, especially methods and agents that do not achieve maximum efficacy in vivo due to the ability of VEGF to prevent their destructive properties. Form a realistic combination.

Thus, the present invention relates to VEGFs that specifically block VEGF binding to the VEGFR2 receptor or that bind essentially only to the VEGFR2 receptor.
Provide an antibody that blocks Such an antibody is known as a VEGFR1 receptor (
VEGF binding to the VEGFR2 receptor (KDR / Flk-1) is significantly inhibited without significantly inhibiting VEGF binding to Flt-1). Therefore,
These antibodies bind to VEGFR2 receptor (KDR / Flk-1)
Inhibits EGF, does not substantially inhibit VEGF binding to the VEGFR1 receptor (Flt-1), exhibits anti-angiogenic and anti-tumor effects in vivo, and has macrophage chemotaxis, osteoclast or cartilage resorption Does not significantly inhibit cell function.

Accordingly, the antibodies of the present invention are referred to simply as "VEGFR2-blocked, non-VEGFR1-blocked, anti-VEGF antibodies." Even more simply, these are "VEGFR
Called "2-blocking anti-VEGF antibodies", which is used for brevity with all compositions, uses and methods of the present invention. "VEGFR2-blocking anti-VEGF antibody" is an antibody to VEGF that blocks VEGF binding to the VEGFR2 receptor. It is clear that such antibodies are not antibodies against the VEGFR2 receptor itself.

Prior to the present invention, there was no motivation to generate anti-VEGF antibodies that specifically block VEGF binding to the VEGFR2 receptor, and no advantages of such antibodies were envisioned. Importantly, the blocking antibody is a growth factor and its receptor (
Because of the need to physically prevent the interaction (s) and the size of the receptor binding site on the growth factor, there was no suggestion that such specific VEGFR2 blocking anti-VEGF antibodies could be developed. .

However, in light of the inventors' surprising discoveries disclosed herein, the art now turns to such specific inhibitory anti-VEGF antibodies, which have different advantages. Provide the knowledge to gain. The present application further provides methodology for generating candidate VEGFR2-blocking anti-VEGF antibodies, and the actual VEG from pools of candidates.
Conventional aspects of the assays required to identify FR2 blocking specific antibodies are described. Thus, in the context of the present invention, a range of VEGFR2 blocking anti-V
Various EGF antibodies are involved in the inhibition of angiogenesis and in the treatment of angiogenic diseases and tumors without inhibiting VEGF signaling via the VEGFR1 receptor and without significant disadvantages and the side effects associated therewith Can be made and used in various embodiments.

As used throughout this specification, the terms “a” and “an” are used to refer to “at least one,” “at least first,” “at least first,” unless the upper limit is specified in detail thereafter. It is used to mean "one or more" or "multiple" reference components or steps. Thus, as used herein, “antibody” means “at least a first antibody”. Possible limitations and parameters of the combination, such as the amount of any single agent, will be known to those of skill in the art in light of the present disclosure.

“VEG binding to VEGF receptor VEGFR2 (KDR / Flk-1)
Antibodies that specifically inhibit F can now be identified by competition and / or functional assays. The preferred assay for simplicity is an ELISA-based competition assay. In a competition assay, VEGF is premixed or mixed with a variable amount of test antibody (eg, 100- to 1000-fold molar excess) and the test antibody is converted to VE
Determine the ability to decrease VEGF binding to GFR2. VEGF can be pre-labeled and detected directly, or detected using (secondary) anti-VEGF antibodies or secondary and tertiary antibody detection systems. The E of such a competitive assay
Although the LISA format is the preferred format, any type of immunocompetition assay can be performed.

[0024] VEGF binding to VEGFR2 in the presence of an irrelevant antibody (including a non-blocking anti-VEGF monoclonal antibody) is a high control (100%) in such a competition assay. In a test assay, a significant decrease in VEGF binding to VEGFR2 in the presence of the test antibody indicates a test compound that significantly inhibits VEGF binding to the VEGF receptor VEGFR2 (KDR / Flk-1).

A significant decrease is “reproducible”, ie, a consistently observed decrease in binding. As used herein, a "significant reduction" refers to at least about 45-fold in any amount between about 100-fold and about 1000-fold molar excess of antibody to VEGF.
%, About 50%, about 55%, about 60%, or about 65% as defined as a reproducible decrease (of VEGF binding to VEGFR2).

A preferred feature of the invention is that the provided antibodies provide VEGF binding to VEGFR1
Are substantially useful, as long as they do not substantially inhibit VEGF binding to VEGFR1, as antibodies that exhibit a moderate significant decrease in VEGF binding to VEGFR2 are still useful. Nevertheless, more preferred antibodies are those that have a more significant ability to inhibit VEGF binding to VEGFR2. These antibodies are at least 70%, about 75%, or about 80% in any amount between about 100-fold and 1000-fold molar excess of the antibody to VEGF
Up to and including reproducible ability to reduce VEGF binding to VEGFR2. It is not necessary to practice the invention, but at least about 85%, about 90%, about 95%
Antibodies that reduce VEGF binding to VEGFR2 by% or even higher are by no means excluded.

Anti-VEGF antibodies, or antigen-binding fragments thereof (these are VEGF
V binding to the VEGF receptor VEGFR2 (KDR / flK-1) without significantly inhibiting VEGF binding to the receptor VEGFR1 (Flt-1)
(Inhibiting EGF) is readily confirmed without VEGFR1 and by a simple competition assay as described above.

[0028] The lack of significant reduction is "reproducible", ie, "substantial maintenance of binding", which is consistently observed. As used herein, "substantially maintaining binding" refers to at least 60%, about 75%, about 80%, Or about 85% (VEGF
Defined as reproducible maintenance (in VEGF binding to R2).

The intention to use an antibody that does not substantially inhibit VEGF binding to VEGFR1 is to maintain the biological function mediated by VEGFR1. Therefore,
The antibody need only maintain VEGF which binds to VEGFR1 sufficient for the biological response to be induced by VEGF. Nevertheless, more preferred antibodies are those that have a more significant ability to maintain VEGF binding to VEGFR1. These antibodies are approximately 100-fold and approximately 1000-fold greater than antibodies to VEGF.
At least 88%, about 90%, about 92% in any amount between fold excess
Binds to VEGFR1 at a level of about 95%, or about 98-99%
Antibodies showing reproducible ability to maintain F.

Antibodies that more substantially inhibit VEGF binding to VEGFR2 are VEGFR
It is understood that a further reduction in the binding of one seems to be acceptable. Similarly, if the antibody has a moderate decrease in VEGF binding to VEGFR2, then VEGFR
Maintaining the bond to one should be pursued more strictly.

The antibody that binds to the VEGF receptor VEGFR2 (KDR / Flk-1)
Another preferred binding assay for identifying and / or confirming that it inhibits EGF is a co-precipitation assay. Co-precipitation assays test the ability of an antibody to block VEGF binding to one or more receptors in solution. In such an assay, VEGF or detectably labeled VEGF is incubated with the appropriate form of the receptor.

There are many ways to perform an immunoprecipitation or co-precipitation assay. In the case of the present invention, the “suitable form of the receptor” is the VEGFR2 in question
It can be a receptor, or the extracellular domain of that receptor. Immunoprecipitation is then carried out, as in the standard reagents, using antibodies against the VEGFR2 receptor, or
Requires the presence of an extracellular domain of the receptor that is different from the site to which F binds.
The present invention provides another "suitable" form of the VEGF receptor, the extracellular domain of the receptor bound to an Fc antibody portion. Such a receptor / F
The c-construct can be precipitated by incubating with a useful immunoprecipitation composition, such as a protein A-based composition.

[0033] Regardless of the appropriate receptor, preferably an immunoprecipitation assay or co-precipitation assay is performed using the control. The ability of only VEGF to bind to the selected receptor should be confirmed by sedimentation in the absence of anti-VEGF antibody. Preferably, parallel incubations are performed in the presence or absence of an antibody with known binding properties that acts as a control. Most preferably, assays using both blocking and non-blocking control antibodies are performed in parallel.

[0034] Any bound immunological species is then immunoprecipitated, for example, by incubation with an effective immunoprecipitation composition (eg, a Protein A composition or Protein A Sepharose beads). The sediment is then tested for the presence of VEGF. If the VEGF in the first incubation was detectably labeled VEGF (eg, radiolabeled VEGF), VEGF in the immunoprecipitate can be detected directly. Any unlabeled VEG in immunoprecipitates
F can be detected by other suitable means, such as by gel separation and immunodetection with anti-VEGF antibodies.

Although the ability of an antibody to block VEGF binding to a VEGF receptor (eg, VEGFR2) can be readily quantified in assays such as co-precipitation assays, the results of the quantification are also informative. Quantification can be achieved by direct measurement of labeled VEGF (ie, for example, by densitometric analysis of immunodetected VEGF). Thus, a reproducible (ie, consistently observed) VE
Antibodies that exhibit the ability to inhibit VEGF binding to GFR2 can be detected, and useful antibodies can be selected according to the quantification criteria outlined above.

Anti-VEGF antibodies that do not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1) are also easily identified by performing a co-precipitation assay as described above using VEGFR1 but not VEGFR2. obtain. Thus, anti-VEGF antibodies that do not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1), but inhibit VEGF binding to the VEGF receptor VEGFR2 (KDR / Flk-1), also provide such a method. And can be easily identified.

The present application also provides various functional assays to identify and / or identify antibodies that significantly inhibit VEGF binding to the VEGF receptor VEGFR2 (KDR / Flk-1). These generally relate to the identification of VEGFR2 as a receptor responsible for certain defined biological responses. Although less preferred than the competitive assay described above, this assay is performed in a cell-free system,
And most reproducible, reliable, labor-saving, and economical, the following assays are nonetheless useful in the context of the present invention.

For example, VEGFR2 blocking anti-VEGF antibodies can be identified by testing for the ability to inhibit VEGF-mediated endothelial cell proliferation (inhibit VEGF mitogenic activity). Any suitable assay can be used with any of a variety of endothelial cells in the presence of VEGF with or without the test antibody. Preferably, duplicate assays are performed in parallel (eg, assays without VEGF and assays with control antibodies of defined characteristics (both blocked and not blocked)). Endothelial cell proliferation is determined and accurately quantified by any acceptable means of determining cell number, preferably including a colorimetric assay.

Antibodies capable of inhibiting VEGF-mediated endothelial cell proliferation are about 25%, 30%
In general, the consistently observed inhibition of VEGF-mediated endothelial cell proliferation by%, 35%, 40%, 45% or 50% or so is shown. Inhibition in such a range indicates an antibody with sufficient characteristics to inhibit angiogenesis in vivo. Antibodies with more significant inhibitory activity are not excluded from the present invention.

Further functional assays for identifying antibodies according to the present invention are assayed to test for blocking VEGF-induced phosphorylation. Any suitable assay can be used with any of a variety of endothelial cells that express any form of native or recombinant phosphorylatable VEGFR2. Cells are incubated with VEGF for the appropriate time in the presence or absence of the antibody to be tested. Preferably, duplicate assays are performed in parallel (eg, assays without VEGF and assays with control antibodies of defined characteristics (both blocked and not blocked)).

[0041] VEGF-induced phosphorylation of VEGFR2 is determined and preferably accurately quantified by any acceptable means. Generally, VEGFR2 is
Immunoprecipitated for further analysis. The degree of phosphorylation of VEGFR2 can be determined directly. For example, the cells can be incubated with ³² P-labeled ATP, which allows for direct quantification of ³² P in immunoprecipitated VEGFR2. Preferably, immunoprecipitated VEGFR2 is obtained by other means (eg,
(By gel separation and immunodetection with antibodies binding to phosphorylated tyrosine residues). Antibodies capable of inhibiting VEGF-induced phosphorylation of VEGFR2 generally show a consistently observed decrease in phosphorylated VEGFR2 levels.

A still further functional assay for identifying VEGFR2-blocking anti-VEGF antibodies according to the invention is assayed to test for inhibition of VEGF-induced vascular permeability. Although any such assay is used, a particular suitable assay is the Miles permeability assay. Here, an animal (eg, guinea pig) is injected with the dye (Evan blue dye), and the appearance of the dye in the animal's skin is determined after the supply of VEGF in the presence or absence of the test antibody. Preferably, duplicate studies are performed in parallel (eg, studies without VEGF and studies with control antibodies of defined characteristics (both blocked and not blocked)). The appearance of the pigment on the animal's skin is typically a spot (eg, a blue spot) on the back of the animal, which can be photographed and measured.

VEGFR2 blocking anti-VEGF antibodies are consistently observed at low concentrations (eg, V
Inhibition of VEGF-induced vascular permeability as inhibition at 100 or 1000 fold molar excess over EGF). V binding to VEGFR2
Antibodies that do not block EGF do not show any significant inhibition of VEGF-induced vascular permeability. Generally, an antibody that blocks VEGF-induced permeability only at high concentrations (eg, 10-fold molarity relative to VEGF) is not an antibody with features according to the invention.

[0044] Broadly acceptable functional assays for angiogenesis factors, and thus anti-angiogenesis factors, are the neoangiogenic corneal micropocket assay and the chicken chorioallantoic membrane assay (CAM) assay. US Pat. No. 5,712,291 is hereby incorporated by reference to show that the corneal micropocket assay and the CAM assay fully predict the identification of factors for use in the very widespread treatment of angiogenic diseases. It is specifically incorporated in the description.

US Pat. No. 5,001,116 is also specifically incorporated herein by reference for the purpose of describing CAM assays. Essentially, fertilized chicken embryos are removed from their hulls on day 3 or 4, and a methylcellulose disc containing the test compound is implanted onto the chorioallantoic membrane. The embryo is examined after about 48 hours, and if a sharp avascular zone appears around the methylcellulose disc, the diameter of this zone is measured. As disclosed in U.S. Patent No. 5,712,291, which is specifically incorporated herein by reference for this purpose in the context of the present invention, the appearance of any avascular zone may include anti-angiogenic factors. Enough to prove. This large area is where the antibody is more effective.

The corneal micropocket assay for angiogenesis can be performed using rat or rabbit corneas. This in vivo model is described in US Pat. No. 5,712,291.
No. 5,871,723, each of which is hereby specifically incorporated by reference herein for the purpose of demonstration, is widely used to make clinical utility predictions. Accepted. Although not believed to be particularly relevant to the present invention, the corneal assay is based on CAM because they generally recognize compounds that are inactive per se, but are metabolized to yield active compounds. Desirable in assays.

In the present invention, this corneal micropocket assay is used to identify anti-angiogenic factors. This is evidenced by a significant reduction in angiogenesis as indicated by a consistently observed and preferably marked decrease in the number of blood vessels in the cornea. Such a response is defined as those corneas that, when contacted with the test substrate, preferably show only spare shoots and / or hairpin loops that show no evidence of sustained growth.

Exemplary VEGFR2 blocking antibodies, anti-VEGF antibodies (and antigen-binding fragments) of the present invention include the following: (a) VE that binds to the VEGF receptor VEGFR2 (KDR / Flk-1)
(B) an antibody that does not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1); (c) inhibits, and preferably significantly inhibits, VEGF-induced phosphorylation of VEGFR2 (D) an antibody that inhibits and preferably significantly inhibits VEGF-induced vascular permeability; (e) an antibody that inhibits and preferably significantly inhibits VEGF-mediated endothelial cell proliferation; B) an antibody that does not significantly inhibit VEGFR1-mediated stimulation or activation of macrophages, osteoclasts or cartilage resorbing cells; and Antibodies that localize to the tumor vasculature and tumor stroma upon administration to animals with metaplastic tumors.

[0049] A particular aspect of the present invention is directed to an antibody that specifically inhibits VEGF binding to the VEGFR2 receptor, has a significant antitumor effect in vivo, and does not inhibit VEGF binding to the VEGFR1 receptor. It is based on the inventors' first surprising findings. In certain embodiments, the present invention thus provides antibodies of defined epitope specificity, wherein such antibodies, or antigen-binding fragments thereof, bind to the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). Essentially combine.

The claimed invention is made possible according to the present specification and makes technical references, know-how, and starting materials readily available. Nevertheless, a sample of a hybridoma cell line producing the 2C3 monoclonal antibody was submitted on March 27, 2000, and published by the American Type Culture Collection (ATCC) (USA Virginia 20110-2209, Manassas, University Boulevard 10801). ) Was deposited as a deposit on March 28, 2000, and assigned ATCC accession number ATCC PTA 1595 on April 11, 2000. This deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure and its provisions (Budapest Treaty). The produced hybridomas are available by the ATCC under the terms of the Budapest Treaty based on the issuance of a U.S. patent along with the associated claims. The availability of this deposited hybridoma should not be construed as a license to practice the invention in contravention of the rights granted under any governmental authority in accordance with that government's patent laws.

Certain preferred compositions are therefore compositions comprising at least a first anti-VEGF antibody, or antigen-binding fragment thereof, or at least a first purified anti-VEGF antibody or antigen-binding fragment thereof (the antigen-binding fragment thereof). Is
A composition comprising at least a first monoclonal antibody, or an antigen-binding fragment thereof, which binds substantially to the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595); ATCC PTA 1595) with the same epitope as VE
And a composition comprising at least a first anti-VEGF monoclonal antibody, or an antigen-binding fragment thereof (these antigen-binding fragments bind to the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595)). is there.

Nevertheless, certain other compositions, antibodies, methods, and in particular, the first and second medical uses of the present invention may be directed to anti-VEGF antibodies, or monoclonal antibody 2C3 (ATCC PTA 1595) itself other than Monoclonal antibody 2C
No. 3 (ATCC PTA 1595) relates to an antigen-binding fragment thereof that binds to the same or substantially the same epitope.

The term monoclonal antibody 2C3 (ATCC PTA 1595) “binds approximately, substantially, or essentially the same, or the same epitope,”
"Cross-reaction" with this monoclonal antibody 2C3 (ATCC PTA 1595)
Antibody. A “cross-reactive antibody” is substantially or essentially identical to monoclonal antibody 2C3 (ATCC PTA 1595), such that it can effectively compete with monoclonal antibody 2C3 (ATCC PTA 1595) for binding to VEGF. Alternatively, an antibody that recognizes, binds to, or has immunospecificity for the same epitope or "epitope site.""2C3 cross-reactive antibodies" are briefly referred to as "2C3-like antibodies" and "2C3-based antibodies", and such terms are used interchangeably herein and refer to compositions, uses and methods. Applied.

The identification of one or more antibodies that bind approximately, substantially, or essentially the same, or the same epitope, as monoclonal antibody 2C3 (ATCC PTA 1595) provides 2C3 with its advantageous characteristics. These are simple technical contents at present. Since the identity of the cross-reactive antibody is determined relative to the reference antibody,
The binding of this reference antibody (2C3) and this test antibody was determined by monoclonal antibody 2C
It is understood that epitopes that are never required to identify antibodies that bind to the same or substantially the same epitope as 3 are actually determined. However, 2C3
Much information on this epitope bound by is contained herein, and epitope mapping is further performed as described by Champe et al. (1995), specifically incorporated herein by reference. Can be done.

The identification of cross-reactive antibodies can be readily determined using any one of a variety of immunological screening assays in which antibody competition can be assessed. All such assays are routine in the art and are described in further detail herein. U.S. Patent No. 5,660,827, issued August 26, 1997, describes a book which relates to a method of making an antibody that binds to the same or substantially the same epitope as a given antibody (eg, 2C3). It is specifically incorporated herein by reference for purposes including further supplementing the teachings.

For example, if the test antibody to be tested is obtained from an animal of a different source or is of an equivalent different isotype, the control (2C3) and the test antibody are mixed (or pre-adsorbed) Alternatively, a sample competition assay can be used and applied to the VEGF antigen composition. By "VEGF antigen composition" is meant any composition comprising a 2C3-binding VEGF antigen described herein (eg,
VEGF is absent). Therefore, protocols based on ELISA and Western blot are suitable for use in such simple competition studies.

In certain embodiments, the change (eg, 1:10 or 1: 100) between the control antibody (2C3) and the test antibody during a period prior to application to the antigen composition.
Pre-mix. In other embodiments, the changes in the control and test antibodies can simply be mixed during exposure to the antigen composition. In any event, by using a species or isotype secondary antibody, only the bound control antibody can be detected, and the binding of the control antibody is reduced by the presence of the test antibody recognizing substantially the same epitope. You.

In conducting antibody competition studies between a control antibody and any test antibody (regardless of species or isotype), a detection label (eg, a biotin or enzyme label (eg, The control (2C3) can be labeled using a radioactive label)). In these cases, the labeled control antibody is pre-mixed or incubated with the test antibody to be tested in various ratios (eg, 1:10 or 1: 100), and (if appropriate after a suitable period of time) and then labeled. The activity of the control antibody is assayed and compared to a potentially competing test antibody using control values not included during the incubation.

The assay may also be any one of a range of immunological assays based on antibody hybridization, and the control antibodies may be detected by means of detecting their label (eg, in the case of biotinylated antibodies). , Using streptavidin, or by using a chromogenic substrate associated with enzyme labeling (eg, 3,3 ', 5,5'-tetramethylbenzine (TMB) substrate with a peroxidase enzyme) or radioactivity. (By simply detecting the label). Antibodies that bind to the same epitope as the control antibody can effectively compete for binding, thus significantly reducing control antibody binding, as evidenced by a decrease in bound label.

The response of this (labeled) control antibody in the absence of a completely irrelevant antibody is a control high. The low value of the control indicates that if competition occurs and the binding of the labeled antibody is reduced, the labeled (2C3) antibody will be of exactly the same type (2
It is obtained by incubating with the unlabeled antibody of C3). In the test assay, a significant decrease in the reactivity of the labeled antibody in the presence of the test antibody is indicative of the test antibody recognizing the same epitope (i.e., the labeled (2C3) antibody and "
Cross-reacting antibodies).

A significant decrease is “reproducible” (ie, a decrease in binding is consistently observed). In the terms of the present invention, a "significant reduction" is at least about 70%, about 75% or about 80% (ELISA) at any ratio between about 1:10 and about 1: 100.
Is defined as a reproducible decrease in 2C3 binding to VEGF in. Antibodies with more stringent cross-blocking activity are about 1:10 and about 1:10
At least about 82%, about 85%, about 88%, about 90%, about 92%, or about 95% or so at any ratio between 0 and 0 (binding to VEGF in an ELISA or other suitable assay) 2C3). Cross-block competition that is not necessary to practice the invention, but shows a reproducible decrease in 2C3 binding to VEGF of about 99%, about 98%, about 97%, or about 96% or so. Or near competition is certainly not ruled out.

The present invention is exemplified by monoclonal antibody 2C3 or
A 1595 is produced by a hybridoma or is an antigen-binding fragment of such a monoclonal antibody. Monoclonal antibody 2C3 (ATC
Hybridomas producing monoclonal anti-VEGF antibodies that bind to substantially the same epitope as C PTA 1595) are another aspect of the invention.

The present invention relates to a method of immunizing an animal with at least a first immunogenic VEGF component, and the method of monoclonal antibody 2C3 (ATCC PTA) from the immunized animal.
1595), wherein the anti-VEGF antibody binds substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595) prepared by a process comprising the step of selecting an antibody that substantially cross-reacts with at least the first antibody; Immunizing an animal with the immunogenic VEGF component and selecting from the immunized animal a cross-reacting anti-VEGF by identifying an antibody that substantially reduces the binding of the 2C3 antibody to VEGF. Further provided is an anti-VEGF antibody that binds substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595) prepared by the following process.

[0064] It binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595) and binds to the VEGF receptor VEGFR2 (KDR / Flk-
An anti-VEGF antibody or an antigen-binding fragment thereof that specifically inhibits VEGF binding to 1); and monoclonal antibody 2C3 (ATCC PTA 1
595) and binds to the same epitope as VEGF receptor VEG
VEG without substantially inhibiting VEGF binding to FR1 (Flt-1)
Anti-VEGF antibodies, or antigen-binding fragments thereof, that inhibit VEGF binding to the F receptor VEGFR2 (KDR / Flk-1) form another aspect of the invention.

Antibodies with such a combination of properties include receptor competition, ELISA,
It can be readily identified by co-precipitation, and / or functional assays, and one or more or a combination of the 2C3 cross-reactivity assays described above. VEGF binding to VEGFR2 is consistently significantly reduced, and VEG binding to VEGFR1
Induction for quantitative evaluation of 2C3-like antibodies that do not consistently significantly inhibit F is as described above.

2C3 measures the amount of VEGF that binds to the VEGFR2 coated ELISA wells.
Approximately 26% each at 100 and 1000 times more moles than VEGF
And a 19% reduction has been shown herein. These numbers equate to about 74% and 81% reduction in VEGF binding to VEGFR2, respectively. 2C3 increased the amount of VEGF bound to the VEGFR2-coated ELISA wells by about 92 and 100 times more than VEGF, respectively, by about 92 times.
% And 105% were indicated herein.

It is further understood that 2C3-like or cross-reactive antibodies that more substantially inhibit VEGF binding to VEGFR2 may have lower resistance in binding to VEGFR1. Similarly, if the antibody has a modest decrease in VEGF binding to VEGFR2, maintenance of binding to VEGFR1 must be more closely followed.

Thus, further exemplary anti-VEGF antibodies (and antigen-binding fragments) of the invention include the following: (a) Non-configurationally independent as assayed by binding to VEGF on a Western blot (B) an antibody that binds to free VEGF; (c) an antibody that binds to VEGF receptor VEGFR2 (KDR / Flk-1)
An antibody that significantly inhibits EGF; (d) an antibody that does not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1); (e) an antibody that inhibits, and preferably significantly inhibits, VEGF-induced phosphorylation of VEGFR2 (F) an antibody that inhibits and preferably significantly inhibits VEGF-induced vascular permeability; (g) an antibody that inhibits and preferably significantly inhibits VEGF-mediated endothelial cell proliferation; (h) inhibits angiogenesis; (I) an antibody that does not significantly inhibit VEGFR1-mediated stimulation or activation of macrophages, osteoclasts or cartilage resorbing cells; (j) tumor blood vessels when administered to an animal with an angiogenic tumor And (k) monoclonal antibody 2C3 (ATCC PTA 159) 5) antibodies that bind to the same or substantially the same epitope.

Compositions, immunoconjugates, medicaments, formulations, cocktails, kits, related to the present invention
In the following description of the first and second medical uses and all methods, the term "antibody"
And "immunoconjugate", or antigen-binding region thereof, refer to VEGFR2 blocking, a range of anti-VEGF antibodies, and specific 2C3 cross-reactive antibodies, unless specifically stated or made clear from scientific terminology.

As used herein, the terms “antibody” and “immunoconjugate” are defined as
Broadly refers to any immunological binding agents, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are assigned to one of five major classes: IgA, IgD, IgE, IgG, and IgM. Some of these are further subdivided into subclasses or isotypes such as, for example, IgG1, IgG2, IgG3, IgG4, and the like. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. This subunit structure and three of the different classes of immunoglobulins
Dimensional structures are well known.

Generally, when antibodies other than the antigen binding region are used in the present invention,
And / or IgM is preferred. Because they are the most common antibodies in physiological conditions, and they are most easily implemented in laboratory settings.

The “light chain” of a mammalian antibody has been assigned to two distinct types: kappa (κ) and lambda (λ), which are based on the amino acid sequence of the constant domain. The use of κ or λ light chains in the antibodies of the present invention is essentially not preferred.

[0073] The use of monoclonal antibodies (MAbs) or derivatives thereof is more preferred. M
Abs are recognized as having certain advantages, such as reproducibility and large-scale production, which are suitable for medical treatment. Accordingly, the present invention provides monoclonal antibodies of the mouse, human, monkey, rat, hamster, rabbit, and even frog or avian species. Mouse, human or humanized monoclonal antibodies are generally preferred.

As will be appreciated by those skilled in the art, immunological binding agents include all antibodies from all species,
And antigen-binding fragments thereof, including dimeric, trimeric and multimeric antibodies; site-specific antibodies; chemical antibodies; human and humanized antibodies; A recombinant antibody; and fragments thereof.

Thus, the term “antibody” is used to refer to any antibody-like molecule that has an antigen-binding region, and the term refers to Fab ′, Fab, F (ab ′) ₂ , a single domain Includes antibody fragments such as antibodies (DAB), Fvs, scFvs (single-chain Fvs), linear antibodies, diabodies, and the like. Techniques for preparing and using various antibody-based structures and fragments are well known in the art (see Kabat et al., 1991, which is specifically incorporated herein by reference). In particular, diabodies are further described in EP 404,097 and WO 93/11161, each of which is specifically incorporated herein by reference. Linear antibodies, on the other hand, are further described in Zapata et al. (1995), which is specifically incorporated herein by reference.

In certain embodiments, a composition of the invention comprises at least a first anti-VEGF antibody, wherein the first anti-VEGF antibody has at least about 75% of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9. , More preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% or at least a first variable region comprising an amino acid sequence region of the same amino acid sequence. Including at least a first variable region. Here, the anti-VEGF antibody is a VEGF of the present invention, as exemplified by the 2C3 antibody.
R2-blocked, retaining at least substantially the biological properties of the anti-VEGF antibody.

The identity or homology with respect to these and other anti-VEGF antibody sequences of the present invention is determined by the sequence of SEQ ID NO: 7 or SEQ ID NO: 9, or another antibody of the present invention after annealing and introducing gaps into the sequence. -Defined herein as the percentage of amino acid residues in the same candidate sequence as the sequence of the VEGF antibody, and achieves, if necessary, the maximum percentage of sequence identity. VEGFR2-block used for sequence comparison,
Maintaining substantially the same, or even more effective, biological properties of the anti-VEGF antibody is of particular importance. Such comparisons are readily performed, for example, using one or more of the various assays described herein.

[0078] In certain preferred embodiments, the anti-VEGF antibodies of the invention comprise SEQ ID NO: 7.
Or at least a first variable region including an amino acid sequence region having the amino acid sequence of SEQ ID NO: 9. This is exemplified by the variable region comprising the amino acid sequence region encoded by the nucleic acid sequence of SEQ ID NO: 6 or SEQ ID NO: 8. Such sequences include CDRs 1-3 (complementary determining regions) of the variable regions of the heavy and light chains,
Sequence of Vh and Vκ of C3ScFv.

In another preferred embodiment, the original VEGFR2-block, anti-VE
Second generation antibodies are provided that have enhanced or superior properties compared to GF antibodies (eg, 2C3). For example, second generation antibodies have stronger binding affinities, more effective blocking of VEGF binding to VEGFR2, more specific blocking of VEGF binding to VEGFR2, weaker blocking of VEGF binding to VEGFR1, VEGF Enhanced ability to inhibit induced endothelial cell proliferation and / or migration, superior ability to inhibit VEGF-induced vascular permeability, and preferably VEGF in vivo, including vascularized tumors It may have increased ability to inhibit induced angiogenesis and treat vascular disease.

[0080] Comparisons to identify effective second generation antibodies are readily processed and quantified using, for example, one or more of the various assays described in detail herein. 2C
VEGFR2-blocked, anti-VEGF of the invention, as exemplified by 3 antibodies
Second generation antibodies having at least about 10-fold, preferably at least 20-fold, and more preferably at least about 50-fold enhanced biological properties or activity compared to the antibody are contemplated by the present invention.

[0081] In certain embodiments, the antibodies used are "humanized", partially human or human antibodies. A "humanized" antibody is a chimeric monoclonal antibody, generally from a mouse, rat or non-human species, having human constant regions and / or various domain domains ("partially human chimeric antibodies"). The various humanized monoclonal antibodies used in the present invention are chimeric antibodies, wherein at least a first antigen binding region, or complementarity determining region (CDR) of a mouse, rat or other non-human monoclonal antibody has Operably linked or "grafted" onto a human antibody constant region or "framework".

A “humanized” monoclonal antibody for use herein can also be a monoclonal antibody from a non-human species, wherein one or more selected amino acids is
It has been replaced by an amino acid more commonly observed in human antibodies. This can be easily achieved by the use of conventional recombination techniques, especially site-directed mutagenesis.

[0083] Fully human antibodies, rather than "humanized", can also be prepared and used in the present invention. Such antibodies can also be obtained from healthy subjects. To accomplish this, one simply obtains a population of mixed peripheral blood lymphocytes from a human subject, including antigen-presenting cells and antibody-producing cells, and divides the cell population into an immunologically effective amount of an aminophospholipid sample. Stimulate in vitro by mixing with Once the human anti-VEGF antibody producing cells are obtained, they are used for hybridoma and / or recombinant antibody production.

A further technique for the production of human monoclonal antibodies involves immunizing a transgenic animal, preferably a transgenic mouse, which comprises
Includes a human antibody library with an immunologically effective amount of VERF sample. This also generates human anti-VEGF antibody producing cells for further manipulation in hybridoma and / or recombinant antibody production, and the advantage that spleen cells rather than peripheral blood cells can be easily obtained from transgenic animals or mice. have.

[0085] VEGFR2 blocking, anti-VEGF antibodies according to the present invention can be readily prepared by processes and methods comprising: (a) preparing candidate antibody-producing cells; and (b) antibodies from candidate antibody-producing cells. In the VEGFR2 (K
Significantly inhibiting VEGF binding to DR / Flk-1) and not significantly inhibiting VEGF binding to VEGF receptor VEGFR1 (Flt-1).

Another antibody according to the invention is monoclonal antibody 2C3 (ATCC PTA
1595) can be readily prepared by selecting an antibody that substantially cross-reacts with 1595). Suitable preparation processes and methods include: (a) preparing candidate antibody-producing cells; and (b) converting antibodies that substantially cross-react with monoclonal antibody 2C3 (ATCC PTA 1595) to candidate antibody-producing cells. The step of selecting from.

One process for preparing suitable antibody-producing cells and obtaining antibodies therefrom is as follows:
It can be performed in situ in a given patient. That is, simply providing a patient with an immunogenically effective amount of an immunogenic VEGF sample will result in appropriate antibody production. Thus, the antibody is still "obtained" from the antibody-producing cells, but is isolated away from the host and does not have to be subsequently provided to the patient, spontaneously localizes to the tumor vasculature, and It can exert its biological antitumor effect. However, such embodiments are not preferred due to the lack of significant specificity.

[0088] Suitable antibody-producing cells can also be obtained, and subsequently, the antibodies can be isolated and / or purified by stimulating peripheral blood lymphocytes with VEGF in vitro.

Another method comprises administering to an animal an immunizing composition comprising at least a first immunogenic VEGF component, and VE binding to VEGFR2 (KDR / Flk-1).
An antibody that significantly inhibits GF and does not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1), and optionally monoclonal antibody 2
Selecting antibodies from the immunized animal for antibodies that substantially cross-react with C3 (ATCC PTA 1595). These methods include the following steps: (a) at least one dose, and optionally one or more doses of an immunologically effective amount of an immunogenic VEGF sample (eg, a first human VEGF component, Immunizing the animal by administering it to a substantially full-length VEGF component, or recombinant human VEGF); and (b) obtaining suitable antibody-producing cells from the immunized animal; For example,
Antibody-producing cells significantly inhibit VEGF binding to VEGFR2 (KDR / Flk-1) and VEG binding to VEGF receptor VEGFR1 (Flt-1).
F that does not significantly inhibit F, and monoclonal antibody 2C3 (A
Producing antibodies that substantially cross-react with TCC PTA 1595).

An immunologically effective amount of a VEGF sample can be administered as a VEGF conjugate or in combination with any suitable adjuvant (eg, Freund's complete adjuvant). Any empirical techniques or modifications can be used to increase immunogenicity. Intact, substantially full-length human VEGF is generally preferred as the immunogen.

[0091] Regardless of the nature of the immunization process or the type of animal being immunized, suitable antibody-producing cells are obtained from the immunized animal and are preferably further manipulated by hand. As used herein, an "immunized animal" is a non-human animal, unless explicitly stated otherwise. Although any antibody producing cells can be used, most preferably, spleen cells are obtained as a source of antibody producing cells. The antibody-producing cells can be used in a preparation process that includes: (a) fusing anti-VEGF antibody-producing cells with immortal cells to prepare a hybridoma that produces a monoclonal antibody according to the present invention; and (b) Obtaining a suitable anti-VEGF antibody from the hybridoma according to the present invention.

The “suitable” anti-VEGF antibody producing cells, hybridomas and antibodies are
Cells, hybridomas and antibodies that produce R2-blocking anti-VEGF antibodies or that exist as VEGFR2-blocking anti-VEGF antibodies, which VEGFR2-blocking anti-VEGF antibodies significantly enhance VEGF binding to VEGFR2 (KDR / Flk-1). And does not significantly inhibit VEGF binding to the VEGF receptor VEGFR1 (Flt-1), and the monoclonal antibody 2
An antibody that substantially cross-reacts with C3 (ATCC PTA 1595).

Thus, methods for preparing monoclonal antibodies based on hybridomas include methods that include: (a) at least one (and optionally more than one) immunologically effective amount of an immunogen. Immunizing the animal by administering a sex VEGF sample (preferably an intact human VEGF sample) to the animal; (b) preparing a collection of monoclonal antibody-producing hybridomas from the immunized animal; (c) At least a first VEGFR2-blocking anti-VEGF antibody according to the present invention;
Optionally selecting from this collection at least a first hybridoma producing an anti-VEGF antibody that substantially cross-reacts with monoclonal antibody 2C3 (ATCC PTA 1595); and (d) at least a first VEGFR2-blocking anti-VEGF antibody. Culturing at least a first antibody-producing hybridoma to provide a VEGF monoclonal antibody; and preferably, (e) culturing the first VEGFR2-
Obtaining a blocked anti-VEGF monoclonal antibody.

In identifying anti-VEGF antibodies that substantially cross-react with monoclonal antibody 2C3 (ATCC PTA 1595), this selection step may include: (a) VEGF samples may be prepared using monoclonal antibody 2C3 (ATCC PTA 1595). 1
595) and contacting with an effective amount of the candidate antibody; and (b) determining the ability of the candidate antibody to substantially reduce binding of the 2C3 antibody to the VEGF sample, wherein the step of 2
The ability of the candidate antibody to substantially reduce the binding of the C3 antibody was determined by monoclonal antibody 2C
3 (ATCC PTA 1595) binds to substantially the same epitope
A step, which is an indicator of a GF antibody.

The selection step may further include: (a) converting the first VEGF to an effective binding amount of monoclonal antibody 2C3 (ATC
(B) contacting the second VEGF with an effective binding amount of the monoclonal antibody 2C3 (ATCC).
Contacting PTA 1595) with an effective competing amount of the candidate antibody, and determining the amount of 2C3 that binds to VEGF in the presence of the candidate antibody; and (c) determining the amount of 2C3 that binds to VEGF. By selecting a candidate antibody that preferably reduces by at least about 80%, the monoclonal antibody 2C3 (ATCC
Identifying an anti-VEGF antibody that binds to substantially the same epitope as PTA 1595).

When non-human animals are used for immunization, the monoclonal antibodies obtained from such hybridomas often have a non-human make up. Such antibodies can optionally be subjected to a humanization process, transplantation or mutation, as known in the art and as further described herein. Alternatively, a transgenic animal containing a human antibody gene library (
Mouse) can be used. Thus, immunization of such animals directly results in the generation of appropriate human antibodies.

After production of suitable antibody-producing cells (most preferably hybridomas), whether producing human or non-human antibodies, the nucleic acid encoding the monoclonal antibody can be a “recombinant” monoclonal antibody. It can be cloned for preparation. Any recombinant cloning technique can be utilized, including the use of PCR ^™ to prime the synthesis of antibody-encoding nucleic acid sequences. Accordingly, still further suitable methods for preparing monoclonal antibodies include those involving the use of antibody-producing cells as follows: (a) at least a first anti-VEGF antibody-producing cell (preferably, a hybridoma); Obtaining a nucleic acid molecule or segment encoding one suitable anti-VEGF antibody; and (b) obtaining said nucleic acid or segment in a recombinant host cell to obtain a recombinant VEGFR2-blocked anti-VEGF monoclonal antibody according to the present invention. A step of expressing

However, other powerful recombinant techniques are available that are ideally suited for the preparation of recombinant monoclonal antibodies. Such recombinant techniques include methods for preparing monoclonal antibodies based on phagemid libraries, including: (a) at least one dose (and optionally more) of the immunological An effective amount of an immunogenic VEGF sample (eg, an intact human VEGF sample)
Immunizing the animal by administering to the animal: (b) RN isolated from antibody-producing cells (preferably spleen) of the immunized animal
Preparing a combinatorial immunoglobulin phagemid library expressing A; (c) at least a first VEGFR2-blocking anti-VEGF antibody, optionally cross-reacting substantially with monoclonal antibody 2C3 (ATCC PTA 1595) Selecting at least a first clone to be expressed from a phagemid library; (d) VEGFR2-blocking anti-VEG from at least the first selected clone
Obtaining a nucleic acid encoding the F antibody; and expressing the nucleic acid in a recombinant host cell to provide at least a first VEGFR2-blocking anti-VEGF antibody; and, preferably, (e) at least the first Obtaining at least a first VEGFR2-blocking anti-VEGF antibody expressed by a nucleic acid obtained from the clone.

Also, in such phagemid library-based techniques, transgenic animals carrying a human antibody gene library can be used to produce recombinant human monoclonal antibodies.

Regardless of the method for preparing the first VEGFR2-blocked anti-VEGF antibody nucleic acid segment, more suitable antibody nucleic acid segments can be readily prepared by standard molecular biology techniques. To confirm that any variant, variant or second generation VEGFR2-blocking anti-VEGF antibody nucleic acid segment is suitable for use in the present invention, the nucleic acid segment may be expressed according to the present invention by expressing a VEGFR2-blocking anti-VEGF antibody. Will be tested to confirm. Preferably, variants, variants or second generation nucleic acid segments are also tested to confirm hybridization under standard, more preferably, standard stringent hybridization conditions. Exemplary suitable hybridization conditions are: about 7% sodium dodecyl sulfate (SDS), about 0.5 M NaPO ₄ , about 1 mM EDTA at about 50 ° C.
And washing with about 1% SDS at about 42 ° C.

A variety of recombinant monoclonal antibodies, whether of human or non-human origin,
As easily prepared, the method of treatment of the present invention may comprise at least a first VEGFR2-blocking anti-VEGF antibody expressing a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody in a patient.
By providing the nucleic acid segment to an animal or patient. "
A nucleic acid segment expressing an antibody based on VEGFR2-blocked, anti-VEGF, 2C3-like or 2C3 "is generally at least in the form of an expression construct, which is comprised in a virus or recombinant host cell. Can be Preferred gene therapy vectors of the invention are generally such as comprised in a recombinant retrovirus, herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV), cytomegalovirus (CMV), and the like. It is a viral vector.

The present invention further provides at least a first purified VEGFR2-blocking anti-VEG
F antibody, or an antigen-binding fragment thereof, if necessary, monoclonal antibody 2
Compositions are provided that include those that bind to essentially the same epitope as C3 (ATCC PTA 1595). Such compositions may be pharmaceutically acceptable compositions for use in laboratory research. In terms of pharmaceutical composition, they can preferably be formulated for parenteral administration, such as intravenous administration.

The present invention relates to VEGFR2-blocking anti-VEGF antibodies (2C3-cross-reactive antibodies,
(Including C3-like or 2C3-based antibodies). For all methods, in the described methods, the terms “a” and “an” are “at least one,” “at least first,” “one or more,” or “
Used to mean "a plurality" of steps (unless specifically stated).
This is particularly relevant for the administration step in the treatment method. Thus, not only can different doses be used in the present invention, but different numbers of doses (eg, injections) can be used, up to and including multiple injections. Combination therapy can be used before, after, or during the administration of the anti-VEGF therapeutic antibody.

A variety of useful in vitro methods and uses having important biological significance are provided. First, there is provided a method of binding VEGF and its use, which generally comprises a composition comprising VEGF, preferably a composition comprising free (non-receptor bound) VEGF. At least a first VEGFR2-blocking anti-V
Effectively contacting the EGF antibody, or an antigen-binding fragment thereof, optionally an antibody that binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595).

Provided are methods of detecting VEGF, and uses thereof, which generally comprise
A composition suspected of containing EGF is treated with at least a first VEGFR2-blocking anti-VE
A GF antibody, or an antigen-binding fragment thereof, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), and conditions effective to permit formation of a VEGF / antibody complex And detecting the complex so formed. The methods and uses of this detection can be used in connection with biological samples, for example, in the diagnosis of angiogenesis and tumors, and diagnostic kits based thereon are also provided.

The present invention provides a method for preferentially or specifically inhibiting VEGF binding to the VEGF receptor VEGFR2 and its use, which generally comprises VEGF
In the presence, a population of cells or tissues comprising endothelial cells expressing VEGFR2 (KDR / Flk-1) is treated with a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody, optionally a monoclonal antibody 2C3 (ATCC PTA
1595), or a composition comprising an antigen-binding fragment thereof, and a VEGF binding to the VEGF receptor VEGFR2
Contacting under conditions effective to inhibit the contact.

Provided are methods of substantially inhibiting VEGF binding to the VEGF receptor VEGFR2, without substantially inhibiting VEGF binding to the VEGF receptor VEGFR1, and uses thereof. These methods involve the use of VGF in the presence of VEGF.
A cell or tissue population comprising a population of endothelial cells expressing EGFR2 (KDR / Flk-1) and VEGFR1 (Flt-1) is treated with a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody, And a composition comprising an anti-VEGF antibody that binds substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or an antigen-binding fragment thereof, and VEGF that binds to VEGF receptor VEGFR1 without significantly inhibiting VEGF Contacting under conditions effective to inhibit VEGF binding to the receptor VEGFR2.

A further method of the invention and its use is in analyzing the biological role of VEGF receptors termed VEGFR2 and VEGFR1,
It involves the following steps: (a) VEGF and VEGFR2 (KDR / Flk-1) and VEGF
A biological composition or tissue comprising a population of cells expressing R1 (Flt-1)
Comprising a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody, optionally an anti-VEGF antibody that binds substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or an antigen-binding fragment thereof. Contacting the composition; and (b) determining the effect of the VEGFR2-blocking anti-VEGF antibody on at least a first biological response to VEGF, wherein: (i) VEGFR2-blocking anti-VEGF Changes in the biological response in the presence of the antibody are indicative of a response mediated by the VEGFR2 receptor; and (ii) maintenance of the biological response in the presence of the VEGFR2-blocking anti-VEGF antibody is indicative of VEGFR1 A process that is indicative of a receptor-mediated response.

Provided are growth inhibition methods and uses, including methods and uses that specifically inhibit VEGF-induced endothelial cell proliferation and / or migration, which generally involve VEGF and a population of endothelial cells. Comprising a population of cells or tissues comprising a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or Contacting a composition comprising an antigen-binding fragment of a VEGFR2-blocking anti-VEGF antibody under conditions effective to inhibit VEGF-induced endothelial cell proliferation and / or migration.

[0110] Methods of using VEGF-induced endothelial cell proliferation and / or migration without significantly inhibiting VEGF-induced macrophage chemotaxis and uses thereof are provided,
This generally results in a biologically effective amount of at least a first VEGFR2-blocking anti-VE
GF antibody, monoclonal antibody 2C3 (ATCC PTA 159
5) a composition that binds to substantially the same epitope as in 5) or an antigen-binding fragment of an anti-VEGF antibody and VEGF-induced endothelial cell proliferation and / or migration without significantly inhibiting VEGF-induced macrophage chemotaxis Contacting under conditions effective to inhibit

A method and use thereof for inhibiting VEGF-induced endothelial cell proliferation and / or migration, and optionally angiogenesis, without significantly inhibiting VEGF stimulation of macrophages, osteoclasts or cartilage resorbent cells Further provided. The method generally comprises treating a population of cells or tissue comprising endothelial cells and at least one of macrophages, osteoclasts or cartilage resorbing cells with a biologically effective amount of at least a first VEGFR2-blocking antibody. VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or a composition comprising an antigen binding fragment of the antibody, and VEGF stimulation of macrophages, osteoclasts or cartilage resorbing cells Contacting under conditions effective to inhibit VEGF-induced endothelial cell proliferation and / or migration, or angiogenesis, without significant inhibition of VGF.

The methods and uses described above can be performed in vitro and in vivo. In the latter case, the tissue or cells are now located in the animal and the anti-VEGF antibody is administered to the animal. In both cases, the methods and uses will be those that inhibit angiogenesis. This causes tissues containing potentially angiogenic blood vessels, or a population of potentially angiogenic blood vessels (ie, those potentially exposed to VEGF) to inhibit angiogenesis. Under effective conditions, the anti-angiogenic composition (a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody (optionally, monoclonal antibody 2C3 (ATCC PTA 1595)) That bind to the same epitope, or an antigen-binding fragment thereof).

If a population of potentially angiogenic blood vessels is maintained ex vivo, the present invention provides
Has utility in drug development programs. In in vitro screening assays, positive positive and negative controls are useful as a first step in the development of drugs that inhibit or promote angiogenesis and in delineating further information on the angiogenic process. When a population of potentially angiogenic blood vessels is placed in an animal or patient, the anti-angiogenic composition is administered to the animal as a form of treatment.

Thus, from each standpoint of the foregoing inhibition methods, a “biologically effective amount” refers to inhibiting VEGF-induced endothelial cell proliferation and / or cell migration; significantly inhibiting VEGF-induced macrophage chemotaxis. Inhibits VEGF-induced endothelial cell proliferation and / or cell migration without inhibition; without significantly inhibiting VEGF stimulation of macrophages, osteoclasts, or cartilage resorbing cells, without inhibiting VEGF-induced endothelial cell proliferation and / or cell migration.
Or VEGFR2, which is effective to attenuate proliferation and / or migration of vascular endothelial cells in a manner effective to inhibit vascular proliferation or angiogenesis; Blocking anti-VEGF antibody (2C if necessary
3-base antibody).

Accordingly, the present invention provides a method of inhibiting VEGF-induced angiogenesis, and its use in inhibiting, and preferably, significantly inhibiting VEGF stimulation of macrophages, osteoclasts, or cartilage resorbing cells. Rather, it provides methods of treating angiogenic diseases and uses in the treatment. The method generally involves contacting a population of cells or tissues, including endothelial cells and at least one macrophage, osteoclast, or cartilage resorbing cell, with the composition. The composition comprises a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody (optionally, substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or antigen binding of the antibody. Fragments are effective in inhibiting VEGF-induced angiogenesis and in treating angiogenic diseases without significantly inhibiting VEGF stimulation of macrophages, osteoclasts, or cartilage resorbing cells Under conditions that bind).

A method for inhibiting VEGF-induced angiogenesis and its use in inhibiting, without causing significant side effects on bone metabolism, and preferably a method for treating and preferably treating an anti-angiogenic disease. Provided. This method is generally
Contacting a tissue or population of angiogenic blood vessels, including vascular endothelial cells and at least one macrophage, osteoclast, or cartilage resorbing cell, with the composition. The composition comprises a biologically effective amount of at least a first VEGFR2-
Blocking anti-VEGF antibody (if necessary, monoclonal antibody 2C3 (ATCC P
TA 1595), or an antigen-binding fragment of this antibody, that is substantially the same as TA 1595), is effective at inhibiting VEGF-induced angiogenesis and significantly impairs the activity of macrophages, osteoclasts, or cartilage resorbing cells. Absent, but which binds under conditions effective to treat an angiogenic disease without producing significant side effects on bone metabolism.

Screening (in vitro) and treating (in vivo) anti-angiogenic drugs may be any disease or disorder characterized by unwanted, inappropriate, abnormal, excessive and / or pathological angiogenesis. Or at risk of developing such a disease. Abnormal angiogenesis occurs in a wide range of diseases and disorders, and certain anti-angiogenic therapies once shown to be effective in any acceptable model system are associated with angiogenesis It is well known to those skilled in the art that it can be used to treat the full range of diseases and disorders that

The methods and uses of the invention are particularly useful for any form of neovascularized tumor; macular degeneration (including age-related macular degeneration); arthritis (including rheumatoid arthritis); atherosclerosis Diabetic retinopathy and other retinopathies; thyroid hyperplasia (including Graves'disease);hemangiomas; neovascular glaucoma; and having or at risk of developing such diseases, Intended for use in animals and patients.

The methods and uses of the present invention may have arteriovenous malformation (AVM), meningioma and vascular restenosis (including restenosis after angioplasty) or the risk of developing such a disease There are further contemplated treatments for animals and patients. Other intended targets for treatment methods and uses include hemangiofibromas, dermatitis, endometriosis, hemophilic joints,
In animals and patients having or at risk for developing hyperplastic scars, inflammatory diseases and disorders, suppurative granulomas, scleroderma, synovitis, trachoma and vascular adhesions is there.

As described in US Pat. No. 5,712,291, each of the somewhat preferred treatment groups described above should never be treated according to the present invention. Is not an exhaustive type of state. US Patent 5,71
No. 2,291 is hereby incorporated by reference for certain specific purposes. The purpose of this is to identify a number of other conditions that can be effectively treated by anti-angiogenic therapy; once a defined category of angiogenesis inhibiting compounds has been disclosed and claimed, all vasculatures The purpose of presenting a unified concept of treatment of plasticizing diseases (
In the case of the present invention, a VEGFR2-blocking anti-VEGF antibody (optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595)); and the treatment of all angiogenic diseases is a single And the purpose of showing that it is possible with the data derived from only the model system.

In a still further aspect, and as described in US Pat. No. 5,712,291, which is incorporated herein by reference, the methods and uses of the present invention provide for the overgrowth of vascular connective tissue, red nose, Acquired immunodeficiency syndrome, arterial occlusion, atopic keratitis, bacterial ulcer, Behcet's disease, blood-born tumor
umor), carotid artery occlusive disease, chemical burn, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitreitis, contact lens exhaustion, corneal graft rejection, corneal neovascularization, corneal graft neovascularization , Crohn's disease, Eels' disease, epidemic keratoconjunctivitis, fungal ulcer, herpes simplex infection, shingles infection, hyperviscosity syndrome, Kaposi's sarcoma, leukemia, lipid degeneration, Lyme disease, marginal keratitis, Mohren's ulcer, leprosy Non-mycobacterial infections, myopia, ocular neovascular diseases, congenital structural defects in the optic disc,
Osler-Weber syndrome (Osler-Weber Rendu), osteoarthritis, Paget's disease, pars planitis, pemphigus, phy
lectenulosis, polyarteritis, post-laser complications, protozoal infection,
Elastic fibrous pseudoxanthoma, pterygium hemikeratosis, radial keratotomy, retinal neovascularization, retinopathy of prematurity, posterior lens fibroplasia, sarcoid, scleritis, sickle cell anemia, Sjogren's syndrome, solid Tumors, Stargardt disease, Steven Johnson disease, upper marginal keratitis, syphilis, systemic lupus erythematosus, terien marginal degeneration, toxoplasmosis, trauma, Ewing sarcoma tumors, neuroblastoma tumors, osteosarcoma tumors, retinoblasts Treating animals and patients with tumors of cell tumors, tumors of rhabdomyosarcoma, ulcerative colitis, venous obstruction, vitamin A deficiency and Wegener's sarcoidosis, or at risk of developing such diseases Intend.

The invention further provides that the subject has or is at risk of developing arteritis.
Methods and uses for the treatment of animals and patients are provided. This is usually due to the treatment of arteritis with immunological factors as described in US Pat. No. 5,753,230, which is hereby incorporated by reference in detail. US Patent 5,972,92
No. 2 is also incorporated by reference herein in detail, including diabetes, parasitic diseases, abnormal wound healing, post-operative hyperplasia, burns, injuries or trauma, impaired hair growth, ovulation and luteinization. Yet further exemplifies the application of anti-angiogenic strategies for the treatment of unwanted angiogenesis associated with inhibition of implantation in the uterus and inhibition of embryonic development. Thus, all of the above conditions are contemplated for treatment by the methods and uses of the present invention.

US Pat. No. 5,639,757, which is incorporated herein by reference in more detail, illustrates the use of anti-angiogenic strategies for the general treatment of graft rejection. Treatment of pulmonary inflammation, nephrotic syndrome, pre-eclampsia, pericardial effusion (including those associated with pericarditis) and pleural effusion using anti-angiogenesis strategies based on VEGF inhibition is described in WO 98/45331 ( Which are hereby incorporated by reference in detail). Accordingly, animals and patients having or at risk of developing any of the aforementioned conditions are contemplated for treatment by the methods and uses of the present invention.

As described in WO 98/16551, which is specifically incorporated herein by reference, biological molecules that antagonize VEGF function are also characterized by unwanted vascular permeability. Suitable for use in treating the diseases and disorders indicated. Thus, for the methods and uses herein, VEGF antagonist antibodies are useful for treating diseases and disorders characterized by unwanted vascular permeability (eg,
Edema associated with brain tumors, ascites associated with malignant tumors, Meggs (Meigs) syndrome, pulmonary inflammation,
(E.g., nephrotic syndrome, endocardial effusion and pleural effusion), or are applicable to the treatment of animals and patients at risk of developing it.

Although treatment of all of the above-mentioned diseases is possible within the integrated invention, particularly preferred aspects of the methods and uses of the invention include the use of vascularized solid, metastatic or primary tumors. Application of anti-angiogenesis therapy to animals and patients who have or are at risk of developing metastases.

A method of inhibiting VEGF-induced angiogenesis and use in this inhibition, and preferably an anti-tumor effect or improved without significantly inhibiting VEGF stimulation of macrophages, osteoclasts, or cartilage resorbing cells Further provided are methods and uses therein that provide an anti-tumor effect. The method generally involves contacting a tissue, tumor environment, or population of angiogenic blood vessels, including vascular endothelial cells and at least one macrophage, osteoclast, or cartilage resorbing cell, with the composition. The composition comprises a biologically effective amount of at least the first VEGF.
R2-blocking anti-VEGF antibody (if necessary, monoclonal antibody 2C3 (ATC
CPTA 1595), or an antigen-binding fragment of this antibody, inhibits VEGF-induced angiogenesis without significantly inhibiting VEGF stimulation of macrophages, osteoclasts, or cartilage resorbing cells Valid for
And under conditions effective to exert an anti-tumor effect or an improved anti-tumor effect,
Binding).

Accordingly, the present invention further provides methods of treating diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis, and uses in this treatment. The method comprises administering to a animal or patient having such a disease or cancer a therapeutically effective amount of at least a first pharmaceutical composition (VEGFR2-blocking anti-VEGF antibody (
Optionally, administering a monoclonal antibody 2C3 (ATCC PTA 1595) that has substantially the same epitope, or that binds to an antigen-binding fragment or immunoconjugate of such a VEGF antibody.

The present invention provides anti-angiogenic methods using unconjugated or naked antibodies and fragments thereof, and vascular targeting methods using immunoconjugates, wherein the antibody or antigen-binding fragment is a therapeutic agent. Operably coupled to both). Thus, unless otherwise specifically stated or made explicit in scientific terminology, as used herein, the term "antibody and fragment thereof"
An "unconjugated or naked" antibody or fragment is meant. It is not conjugated to another agent, especially a therapeutic or diagnostic agent. These definitions refer to modifications of the antibody (for example, but merely by way of example, that alter the biological half-life, affinity, avidity, or other properties of the antibody, or that the antibody and other effectors ) Is not excluded.

The methods and uses of the anti-angiogenic treatment of the present invention also encompass the use of both unconjugated or naked antibodies and immunoconjugates. In immunoconjugate-based anti-angiogenic treatment methods, the antibody or antigen-binding fragment thereof is preferably a second anti-angiogenic factor (the first anti-angiogenic factor, the anti-VEGF antibody itself). Operatively connected to the The bound anti-angiogenic factor can be a factor having a direct or indirect anti-angiogenic effect.

[0130] The anti-angiogenic treatment methods and uses may be used in animals or patients with diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis, in a therapeutically effective amount. Administering at least a first pharmaceutical composition of The pharmaceutical composition comprises an unconjugated or naked VEGFR2-blocking anti-VEGF antibody, or an antigen-binding fragment thereof (optionally, monoclonal antibody 2C3
(Binding to substantially the same epitope as ATCC PTA 1595). Similarly, an administered antibody can be operably associated with a second anti-angiogenic factor.

[0131] Methods for treating metastatic cancer, and uses in the treatment, involve administering to animals and patients with metastatic cancer a therapeutically effective amount of at least one pharmaceutical composition. I do. The pharmaceutical composition comprises at least a first unconjugated or naked VEGFR2-blocking anti-VEGF antibody, or an antigen-binding fragment thereof (optionally, monoclonal antibody 2C3 (ATCC P
TA 1595)). A further method is a method wherein the administered antibody can be operably linked to a second anti-angiogenic factor.

The methods for reducing metastasis from a primary cancer and uses in reducing the same comprise administering to animals and patients having or treating a primary cancer at least one therapeutically effective amount. Administering one first unconjugated or naked VEGFR2-blocking anti-VEGF antibody, or antigen-binding fragment thereof; wherein the unconjugated or naked VE
The GFR2-blocking anti-VEGF antibody, or antigen-binding fragment thereof, optionally binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). Similarly, an administered antibody can be operably associated with a second anti-angiogenic factor.

Diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis
The method for treating and the use in treatment thereof comprises administering to an animal or patient having such a disease (eg, a vascularized tumor) at least a first unconjugated or naked VEGFR2-block. An anti-VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595) or an antigen-binding fragment thereof, is used to induce angiogenesis at the site of the disease or neovascular tumors. The method further comprises the step of administering an amount effective to inhibit. Similarly, an administered antibody can be operably associated with a second anti-angiogenic factor.

Diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis
For use in treating and treating animals or patients with such a disease or cancer, comprising administering to at least a first unconjugated or naked VEGFR2-blocking anti-VEGF antibody, Depending on the monoclonal antibody 2C3 (ATCC PTA 1595), or an antigen-binding fragment thereof, that inhibits VEGF from binding to the VEGF receptor VEGFR2 (KDR / Flk-1). Thus, further comprising administering an amount effective to inhibit angiogenesis at the site of the disease or cancer. The administered antibody may alternatively be operably associated with a second anti-angiogenic factor.

Diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis
The method for treating and the use in the treatment also comprises treating an animal or patient with an angiogenic tumor with a therapeutically effective amount of at least a first unconjugated or naked VEGFR2-blocking antibody. Administering a VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595) or an antigen-binding fragment thereof; wherein the anti-VEGF antibody comprises: VEGF is the VEGF receptor VEGFR1 (
VEGF substantially inhibits binding to the VEGF receptor VEGFR2 (KDR / Flk-1) without significantly inhibiting binding to Flt-1).
Similarly, an administered antibody can be operably associated with a second anti-angiogenic factor.

Diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis
A still further method for treating, and use in, treating an animal or patient having such a disease, cancer or angiogenic tumor in a therapeutically effective amount of at least a first unconjugated Or naked VEGFR2-blocking anti-VEGF antibody, (optionally monoclonal antibody 2C3 (ATCC PTA
1595), wherein the anti-VEGF antibody significantly binds VEGF to the VEGF receptor VEGFR1 (Flt-1). Without inhibiting VEGF, the VEGF receptor VEGFR2 (KDR / Flk-1)
Substantially inhibits angiogenesis in the site of disease, cancer or angiogenic tumor without significantly impairing macrophage chemotaxis in the animal. The administered antibody can be operably associated with the second anti-angiogenic factor.

Diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis
A still further method for treating, and use in, treating an animal or patient having such a disease, cancer or angiogenic tumor, is not a therapeutically effective amount of at least a first conjugate. Or bare VEGFR2-blocking anti-VE
GF antibody, (if necessary, monoclonal antibody 2C3 (ATCC PTA 1
595), which binds to substantially the same epitope or antigen-binding fragment thereof); wherein the anti-VEGF antibody comprises VEGF
Without significantly inhibiting binding to the GF receptor VEGFR1 (Flt-1), VEGF substantially inhibits binding to the VEGF receptor VEGFR2 (KDR / Flk-1), thereby resulting in macrophages, destruction in animals. Inhibits angiogenesis in disease sites, cancer or neovascular tumors without significantly inhibiting the activity of bone cells and / or cartilage resorbing cells. Similarly, the administered antibody
It can be operatively associated with a second anti-angiogenic factor.

[0138] Methods for treating diseases associated with angiogenesis, including all forms of cancer associated with angiogenesis, and use in treating the same, include the use of such diseases (eg, vascular An animal or patient with a neoplastic tumor) at least a first unconjugated or naked VEGFR2 blocking anti-VEGF antibody, optionally, substantially the same epitope as monoclonal antibody 23 (ATCC PTA 1595) Or an antigen-binding fragment thereof is administered in an amount effective to inhibit angiogenesis at the site of the disease or in the vascularized tumor without significantly affecting bone metabolism. The method further includes the step of:

The methods and uses of the anti-angiogenic treatments described above generally involve administering to a mammal or patient systemically (eg, by percutaneous injection, intramuscular injection, intravenous injection, etc.) a pharmaceutically active agent. Administering the composition. However, any route of administration that allows for the localization of the therapeutic agent at the site (s) of angiogenesis, including the tumor or vascular endothelial cells within the tumor, is acceptable. Thus, other suitable routes of delivery include oral, rectal, nasal, topical, vaginal. US Patent 5,7
12,291 is specifically incorporated herein by reference for further description of various routes of administration that may be included in combination with the treatment of an angiogenic disease or disorder.

For use and methods for treating arthritis, for example, as described for other immunological agents in US Pat. No. 5,753,230, which is specifically incorporated herein by reference, Intrabursal administration may be used. For conditions relating to the eye, ophthalmic prescription and administration are contemplated.

“Administration” as used herein refers to an amount and time of a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic agent effective to exert an anti-angiogenic and / or anti-tumor effect. Means providing or delivering. Passive administration of proteinaceous therapeutics is generally preferred, in part, for its simplicity and reproducibility.

However, the term “administering” is used herein to refer to VEGFR2 blocking anti-VEGF
It is used to refer to any and all means by which the antibody or 2C3-based therapeutic is delivered to the tumor vasculature or otherwise provided. Therefore, "administration"
This includes providing cells that produce a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic in a manner effective to effect delivery to the tumor. In such embodiments, it may be desirable to formulate or package the cells into a selectively permeable membrane, structure or implantable device, generally that can be removed to interrupt treatment. Exogenous VEGFR2 blocking anti-VEGF antibody or 2C3-like administration is more generally preferred, which represents a non-invasive method that allows the dose to be closely monitored and controlled.

The therapeutic methods and uses of the present invention also provide a method for converting VEGFR2-blocking anti-VEGF antibodies or 2C3-based therapeutics to effect their expression in the vicinity of or at the location of a tumor. Extending to providing nucleic acids that encode in a fashion. Any gene therapy technique, including naked DNA delivery, recombinant genes and vectors, cell-based delivery, including ex vivo manipulation of patient cells, etc., can be used.

In yet a further embodiment, the present invention provides methods for and in delivering a selected therapeutic or diagnostic agent to blood vessels in angiogenesis associated with a disease. Such embodiments are preferably used to deliver a selected therapeutic or diagnostic agent also to the tumor or intratumoral vasculature or stroma,
The diagnostic or therapeutic agent is then operable to bind to a VEGFR2-blocking anti-VEGF antibody or antigen-binding fragment thereof, optionally one that binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA1595). Administering a biologically effective amount of the composition comprising at least the first immunoconjugate to be bound to a mammal or patient having a vascularized tumor.

Although understanding the mechanism of action underlying the targeting aspects of the present invention is not required to practice such embodiments, the antibodies of the present invention may be associated with VEGFR1 expressed on them. It is believed that binding the bound VEGF will deliver the bound agent to the angiogenesis and tumor vasculature. Accordingly, these methods and uses of the present invention relate to delivering selected therapeutic or diagnostic agents to angiogenic blood vessels, tumors, or intratumoral vasculature, and At least a first VEGFR2 blocking anti-VEGF in a manner effective to allow the antibody to bind to VEGF bound to VEGFR1 that is expressed, overexpressed, or upregulated in the vasculature Antibodies or their antigen-binding fragments, optionally monoclonal antibody 2C3 (ATCC PT
A 1595) binds to substantially the same epitope as the immunologically effective amount of the composition comprising an immunoconjugate to which a diagnostic or therapeutic agent is operably linked. Administering to a mammal or patient in need thereof, ie, delivering a diagnostic or therapeutic agent to VEGF-VEGFR1 in the angiogenic blood vessels, tumor or intratumoral vasculature.

Delivery of a selected therapeutic agent to a tumor or intratumoral vasculature or stroma may be used to stop blood flow, or particularly to stop blood flow within the tumor vasculature; It acts in particular to destroy tumor vasculature; and to induce necrosis, or to induce specific necrosis in tumors. Thus, these methods and uses can be summarized as methods for treating a mammal or patient having an angiogenic tumor, the method comprising operably binding a VEG to a therapeutic agent.
FR2 blocking anti-VEGF antibody, monoclonal antibody 2C3 (ATCC
A therapeutically effective amount of at least a first pharmaceutical composition, comprising at least a first immunoconjugate comprising one that binds substantially the same epitope as PTA 1595), or an antigen-binding fragment thereof. Administering to a mammal or a patient.

A “therapeutically effective amount” for use in the present invention is to specifically kill at least some tumors or vascular endothelial cells within tumors; at least some tumors or intratumorally. To specifically induce cell death in vascular endothelial cells; to specifically promote coagulation in at least some tumors or intratumoral blood vessels; Effective to destroy; specifically to induce necrosis within at least some tumors; and / or to induce tumor regression or remission upon administration to a selected mammal or patient. Nah, VEG
The amount of FR2 blocking anti-VEGF antibody or 2C3-based immunoconjugate. While such an effect is achieved, there is little or no binding to vascular endothelial cells in normal healthy tissue or little or no killing of vascular endothelial cells in normal healthy tissue Indicating little or no coagulation upon occlusion or destruction of blood vessels in healthy normal tissue; and negligible or manageable adverse side effects on normal healthy tissue in mammals or patients. Show.

Thus, in the context of promoting coagulation within the tumor vasculature, or destroying the tumor vasculature, and / or binding to tumor correlates, and / or causing tumor necrosis herein, The terms "preferably" and "especially" as used in refer to the binding of a VEGFR2-blocking anti-VEGF antibody or 2C3-based immunoconjugate to a stroma that is substantially restricted to the tumor stroma, vasculature and tumor site, It is meant to function to achieve coagulation, destruction, and / or tumor necrosis, and does not substantially extend to coagulation, destruction, and / or tissue necrosis in a mammal or subject's normal healthy tissue. Thus, the structure and function of healthy cells and tissues is maintained substantially unimpaired by the practice of the present invention.

While the antibodies of the present invention effectively deliver drugs to the angiogenesis and tumor vasculature by binding to VEGF associated with VEGFR1, other methods and uses have led to the delivery of therapeutic agents to tumor stroma. It operates on the basis of delivering, where it exerts a therapeutic effect on nearby blood vessels. These methods and uses comprise the use of at least a first amount of an immunoconjugate in an amount effective to bind non-receptor-bound VEGF in the tumor stroma.
Comprising a therapeutic agent operably linked to a VEGFR2 blocking anti-VEGF antibody, or an antigen-binding fragment thereof, optionally those that bind substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). Administering the conjugate to a mammal or patient having an angiogenic tumor.

These methods and uses may comprise at least a first VEGFR2-blocking anti-VEGF antibody, or an antigen-binding fragment thereof, in an amount effective to localize the immunoconjugate within the tumor stroma. Monoclonal antibody 2C3 (ATCC PTA
An immunoconjugate comprising a therapeutic agent operably linked to one that binds to substantially the same epitope as 1595) is administered to a mammal or patient having an angiogenic tumor, such that the conjugated therapeutic is administered. The pharmaceutical agent exerts an antitumor effect on surrounding tumor vasculature and / or tumor cells.

The compositions and methods and uses of the present invention may comprise at least a first VEGFR2 blocking anti-V operably linked to at least a first therapeutic or diagnostic agent.
A composition comprising a VEGFR2-blocking anti-VEGF antibody or a 2C3-based immunoconjugate comprising an EGF antibody, or an antigen-binding fragment thereof, optionally one that binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). Extend to things. The VEGFR2 blocking anti-VEGF antibody or 2C3-based therapeutic conjugate is preferably a radiation therapy agent, an anti-angiogenic agent, a cell death-inducing agent, an anti-tubulin agent, an anti-cellular or cytotoxic agent, or a coagulant. (Coagulation factor).

Thus, the present invention provides a range of conjugated antibodies and fragments thereof, wherein the antibodies are operably linked to at least a first therapeutic or diagnostic agent. The term "immunoconjugate" is widely used to define an effective binding of an antibody to another active agent, and is not intended to refer to any type of effective binding exclusively, and is not specifically intended to refer to chemical binding. It is not limited to “conjugation”. The recombinant fusion protein
Special consideration is given. As long as the delivery or targeting agent can bind to the target and the therapeutic or diagnostic agent is sufficiently functional upon delivery, the mode of attachment is appropriate.

[0153] Binding of agents through the carbohydrate moiety of the antibody is also contemplated. Glycation (both O- and N-linked) occurs naturally within the antibody. Recombinant antibodies are modified, if desired, to regenerate or create additional glycation sites, which may include the appropriate amino acid sequence (Asn-X-Ser, Asn-X-Thr, Ser, or Thr). It is easily achieved by engineering into the primary sequence of the antibody.

Use of a VEGFR2 blocking anti-VEGF antibody or 2C3-based therapeutic conjugate,
Presently preferred agents for and related methods and uses are those agents that supplement or enhance the effects of the antibody and / or the antibody selected for the particular tumor type or patient. As "therapeutic agent that supplements or enhances the effects of the antibody", a radiation therapy agent, an anti-angiogenic agent, a cell death-inducing agent and an anti-tubulin agent, any one of which is preferred for use herein These drugs are mentioned.

Attachment or association of a preferred agent with a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody provides an “immunoconjugate,” where such immunoconjugates are often enhanced. It also has more synergistic anti-tumor properties. Currently preferred anti-angiogenic agents for use in this manner are angiostatin, endostatin, angiopoietin.
n), vasculostatin, vasstatin (ca)
nstatin) and maspin. Currently preferred anti-tubulin drugs include colchicine, taxol, vinblastine, vincristine, vindestine and combretastatin (combretastatin).
(statin).

The use of anti-cellular and cytotoxic agents produces VEGFR2-blocking anti-VEGF antibodies or 2C3-based “immunotoxins”, while the use of coagulation factors
GFR2-blocking anti-VEGF antibody or 2C3-based "coag ligand (coa
guligand) ". At least two therapeutic agents (eg, one or more radiation therapy agents, anti-angiogenic agents, cell death-inducing agents, anti-tubulin agents,
Combinations of anticellular and cytotoxic agents and clotting factors) are also contemplated.

In certain applications, a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic is a cytotoxic agent that has the ability to kill endothelial cells or inhibit endothelial cell proliferation or cell division, Operably linked to a drug or other anticellular drug. Suitable anticellular agents include chemotherapeutic agents, as well as cytotoxins and cytostatic agents. Cytostatic agents are generally agents that disrupt the natural cell circulation of the target cell, preferably resulting in the cell being removed from the cell circulation.

Representative chemotherapeutic agents include steroids; cytokines; anti-metabolites (eg, cytokines arabinoside, fluorouracil, methotrexate or aminopterin); anthracyclines; mitomycin C; vinca alkaloids;
Antibiotics; demecortin; etoposide; mitramycin; and antitumor alkylating agents (eg, chlorambucil or melphalan). actually,
Any of the agents disclosed in Table C herein can be used. Certain preferred anti-cellular agents are DNA synthesis inhibitors (eg, daunorubicin, doxorubicin, adriamycin, etc.).

In certain therapeutic applications, toxin moieties are preferred due to the much greater ability of most toxins to deliver cell-killing effects as compared to other potential agents. Accordingly, certain preferred anti-cellular agents for VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibody constructs are plant-derived toxins, fungal-derived toxins, or bacterial-derived toxins. As a representative toxin, epipodophyl toxin (epipodophyll)
llotoxin); bacterial endotoxin or the lipid A portion of bacterial endotoxin; ribosome-inactivating proteins (eg, saporin or gelonin)
gelonin)); α-sartin (α-sarcin);
spergillin; restrictocin; ribonucleases (eg, placental ribonuclease); diphtheria toxin and pseudomonas exotoxin.

A preferred toxin is an A-chain toxin (eg, ricin A-chain). The most preferred toxin moiety is often ricin A, which has been treated to modify or remove carbohydrate residues, and is referred to as "deglycosylated A chain (dgA)." Deglycosylated ricin A chain is preferred because of its extreme potency, long lifespan, and because it is economically easy to manufacture in clinical grade and scale. Recombinant ricin A chains and / or truncated ricin A chains can also be used.

The following patents and patent applications for tumor targeting and treatment with immunotoxins are incorporated herein by reference, in particular to further supplement the present teachings on anti-cellular and cytotoxic agents. Incorporated as: US application Ser. No. 07 / 846,3.
08 / 295,868 (U.S. Pat. No. 6,004,554); 08 / 205,330 (U.S. Pat. No. 5,855,866); 08/35/08
No. 0,212 (U.S. Pat. No. 5,965,132);
No. 08 / 457,487 (U.S. Pat. No. 5,863,538); No. 08 / 457,229 and No. 08/4.
57,031 (U.S. Pat. No. 5,660,827) and 08 / 457,8.
No. 69 (US Pat. No. 6,051,230).

2C3-Based Anti-VEGF Antibodies or Other VEGFR2-Blocking Anti-VEGs of the Invention
The F antibody can be linked to an anti-tubulin drug. As used herein, an “anti-tubulin drug” preferably directly or indirectly inhibits tubulin activity (preferably tubulin polymerization or tubulin depolymerization) required for cell mitosis. By doing so is meant any agent, drug, prodrug or combination thereof that inhibits cell mitosis.

Currently preferred anti-tubulin drugs for use herein are colchicine; taxanes (eg, taxol); vinca alkaloids (eg, vinblastine, vincristine and vindesine); and combretastatin (com
breastatin). Representative combretastatins are combretastatins A, B and / or D (A-1, A-2, A-3, A-4, A-5,
A-6, B-1, B-2, B-3, B-4, D-1 and D-2) and their prodrug forms.

A VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic can include a component that can promote coagulation (ie, a coagulant). Here, the targeting antibody can be directly or indirectly linked to an agent that stimulates coagulation directly or indirectly, for example, via another antibody.

Preferred clotting factors for such uses are Tissue Factor (
TF) and TF derivatives (eg, truncated TF (tTF), dimerized TF, trimerized TF, polymerized TF / multimerized TF, and mutations lacking the ability to activate factor VII Body TF). Other suitable coagulation factors include vitamin K-dependent coagulants (eg, Factor II / IIa, Factor VII / VIIa, IX / IXa
Factor and factor X / Xa); a vitamin K-dependent coagulation factor lacking Gla modification; Ru
ssell viper venom factor X activators; platelet activating compounds (eg, thromboxane A ₂ and thromboxane A ₂ synthase); and inhibitors of fibrinolysis (eg, α2-antiplasmin).

Tumor targeting and treatment with coag ligands are described in the following patents and patent applications, each of which is incorporated herein by reference to yet further augment the present teachings, particularly with respect to coag ligands and coagulation factors: No. 07 / 846,349; 08 / 205,330 (U.S. Pat.
No. 866); 08 / 350,212 (U.S. Pat. No. 5,965,132).
08 / 273,567; 08 / 482,369 (U.S. Pat.
___, ___; payment of patent issuance fee on October 20, 1998);
No. 485,482; 08 / 487,427 (U.S. Pat. No. 6,004,55).
No. 5); 08 / 479,733 (U.S. Pat. No. 5,877,289);
And 08 / 472,631; 08 / 479,727 and 08
/ 481,904 (U.S. Patent No. 6,036,955).

The preparation of immunoconjugates and immunotoxins is generally well known in the art (
For example, US Pat. No. 4,340,53, which is incorporated herein by reference.
No. 5). The following patents and patent applications are further incorporated herein by reference to yet further supplement the present teachings on the generation, purification and use of immunotoxins: US Application No. 07 / 846,349; 08 / 295,868
(U.S. Pat. No. 6,004,554); 08 / 205,330 (U.S. Pat. No. 5,855,866); and 08 / 350,212 (U.S. Pat.
08 / 456,495 (U.S. Pat. No. 5,776,427)
No. 08 / 457,487 (U.S. Pat. No. 5,863,538); No. 08 / 457,229 and No. 08 / 457,031 (U.S. Pat. No. 5,663)
No. 0,827) and 08 / 457,869 (U.S. Pat.
No. 30).

Advantages can be achieved through the use of specific linkers in the preparation of immunoconjugates and immunotoxins. For example, linkers containing sterically "hindered" disulfide bonds are preferred because of their greater stability in vivo and thus prevent release of the toxin moiety prior to attachment at the site of action. .
In general, conjugates that remain intact under the conditions found anywhere in the body except for the intended site of action (in this regard, it is desired that the conjugate have good "release" properties) It is desired to have.

Depending on the particular toxin compound used, it may be necessary to provide a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody and a peptide spacer that operably attaches the toxin compound. Here, this peptide spacer is
It can be folded into a disulfide-bonded loop structure. Proteolytic cleavage within the loop then produces a heterodimeric polypeptide, wherein the antibody and toxin compound are joined by only a single disulfide bond.

When certain other toxin compounds are utilized, the non-cleavable peptide spacer is
An EGFR2-blocking anti-VEGF antibody or 2C3-based antibody and a toxin compound can be provided to operably adhere. Toxins that can be used with non-cleavable peptide spacers are those that can themselves be converted to a cytotoxic disulfide-bonded form by proteolytic cleavage. Examples of such toxin compounds include P
pseudomonas exotoxin compound.

A variety of chemotherapeutic agents and other drugs can also be successfully conjugated to VEGFR2-blocking anti-VEGF antibodies or 2C3-based therapies. Exemplary antineoplastic agents conjugated to the antibody include doxorubicin, daunomycin, methotrexate and vinblastine. In addition, other drugs (eg, neocarzinostatin, macromycin, trenimon (t
renimon) and α-amanitin) have been described (U.S. Patent Nos. 5,660,827; 5,855,866; and 5,965,1).
32, each of which is incorporated herein).

In light of one of our previous studies, coag ligands (coagu
preparation is also easily performed here. VEGFR2 blocking anti-V
Operable association of one or more clotting factors with an EGF antibody or a 2C3-based antibody can be a direct linkage (such as the linkage described above for immunotoxins). Alternatively, the operable association is an indirect attachment (eg, where the antibody is operably attached to a second binding region, preferably an antibody or antigen binding region of an antibody that binds a coagulation factor). obtain. The coagulation factor should be attached to the VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody at a site different from the functional coagulation site, especially if a covalent bond is used to attach the molecule.

Indirectly bound coag ligands are often based on bispecific antibodies. Preparation of bispecific antibodies is also well known in the art. One method of preparation involves the separate preparation of antibodies that have specificity for the targeted tumor component on the one hand and specificity for the coagulant on the other. A pepsin F (ab'γ) ₂ fragment from the two selected antibodies is then generated, and then the separated Fab'γS
Each is reduced to provide an H fragment. The SH group for one of the two partners to be coupled is then alkylated using a cross-linking reagent (eg, o-phenylenedimaleimide) to provide a free maleimide group on one partner. . This partner can then be conjugated to another by a thioether bond to give the desired F (ab'γ) ₂ heteroconjugate (G
Lennie et al., 1987; incorporated herein by reference). Other approaches, such as cross-linking with SPDP or Protein A, can also be implemented.

Another method for generating bispecific antibodies fuses two hybridomas to form a quadroma. As used herein, the term "quadroma" is used to describe a productive fusion of two B cell hybridomas. Using this standard technique, two antibodies that produce hybridomas are fused to give daughter cells, and then cells that maintain expression of both sets of clonotype immunoglobulin genes are selected.

A preferred method of producing quadromas involves the selection of at least one enzyme deficient mutation of the parent hybridoma. This first mutant hybridoma cell line is then fused to a cell of a second hybridoma that has been lethally exposed, for example, to iodoacetamide, which prevents its continued survival. Cell fusion allows the rescue of a first hybridoma by obtaining a gene for its enzyme deficiency from a lethally processed hybridoma, and a second hybridoma via fusion to the first hybridoma. To enable rescue. Fusion of immunoglobulins of the same isotype but different subclasses is preferred but not required. Mixed subclass antibodies may be used in the case of an alternative assay for the isolation of the preferred quadromas.

Microtiter identification embodiments, FACS, immunofluorescent staining, idiotype-specific antibodies, antigen binding competition assays and other methods common in the field of antibody characterization can be used to identify preferred quadromas . After quadroma isolation,
Bispecific antibodies are purified away from other cellular products. This can be accomplished by various antibody isolation procedures known to those skilled in the art of immunoglobulin purification (
For example, Antibodies: A Laboratory Manual, 1
988; incorporated herein by reference). A Protein A Sepharose column or a Protein G Sepharose column is preferred.

In the preparation of immunoconjugates, immunotoxins and coag ligands, recombinant expression may be utilized. Selected VEGFR2 blocking anti-VEGF antibody or 2C3-
The base antibody and the nucleic acid sequence encoding the therapeutic, toxin or coagulant are attached in-frame in the expression vector. Thus, recombinant expression results in translation of the nucleic acid, producing the desired immunoconjugate. Chemical crosslinkers and avidin: biotin crosslinks can also bind therapeutic agents to VEGFR2 blocking anti-VEGF antibodies or 2C3-based antibodies.

The following patents and patent applications are each incorporated herein by reference for the purpose of further supplementing the present teachings on coag ligand preparation, purification and use, including bispecific antibody coag ligands. U.S. Application No. 07/846,
349; 08 / 205,330 (U.S. Pat. No. 5,855,866); 08/3.
50,212 (US Pat. No. 5,965,132); 08 / 273,567; 0
8 / 482,369 (U.S. Pat. No. 5, _, _; paid patent issuance fee on October 20, 1998); 08 / 485,482; 08 / 487,427 (U.S. Pat. No. 6,004) 08 / 479,733 (U.S. Patent No. 5,877).
08 / 472,631; and 08 / 479,727 and 08.
/ 481,904 (U.S. Patent No. 6,036,955).

Immunoconjugates with radiotherapeutic agents, anti-angiogenic factors, apoptosis inducers, anti-tubulin drugs, toxins and coagulants (whether prepared by chemical conjugates or prepared by recombinant expression) Anyway) may utilize a biologically releasable bond and / or a selectively cleavable spacer or linker. Such compositions are preferably reasonably stable during circulation and are released preferentially or specifically upon delivery to a disease or tumor site.

A particular preferred example is an acid-sensitive spacer, wherein VEGFR2 blocking anti-VEGF antibodies conjugated to colchicine or doxorubicin are specifically contemplated.
Other preferred examples are peptide linkers containing cleavage sites for peptidases and / or proteinases that are present specifically or preferentially or are active in the disease site (eg, tumor environment). Delivery of the immunoconjugate to a disease or tumor site results in cleavage of the coagulation factor and relatively specific release of the coagulation factor.

Urokinase, prourokinase, plasmin, plasminogen, TGFβ
, Staphylokinase, thrombin, IX
Peptide linkers containing cleavage sites for factor a, factor Xa or metalloproteinases (MMPs) (eg, stromal collagenase, gelatinase or stromelysin) are described in US Pat. No. 5,877,289 (herein incorporated by reference). As described in Table B2 herein, and further exemplified in Table B2 herein, and enabled by this US Patent No. 5,877,289, for such purposes, Particularly preferred.

VEGFR2 blocking anti-VEGF antibodies may also be derivatized to introduce functional groups,
It may allow for attachment of the therapeutic agent via a biologically releasable bond. Thus, the targeted antibody can be derivatized to introduce side chains and terminate hydrazide, hydrazine, primary or secondary amine groups. Therapeutic agents can be conjugated via a Schiff base bond, a hydrazone or an acyl hydrazone bond or a hydrazide linker (US Pat. Nos. 5,474,765 and 5,762,918, each of which is incorporated herein by reference). Which are specifically referred to as references).

[0183] Whether inherently anti-angiogenic or vascular targeting, the compositions and methods of the present invention can be used in combination with other therapeutic and diagnostic methods. In accordance with the present invention, the term "combined" in terms of a biological agent (preferably a diagnostic or therapeutic agent) (eg, a 2C3-based antibody) for use in "combination" with a VEGFR2-blocking anti-VEGF antibody , Used concisely and span the scope of the embodiments. The term “combined” is clear from scientific terms, unless otherwise specified, and thus refers to the combined compositions, medicaments, cocktails, kits, methods, and various forms of the first and second medical uses. Apply to type.

Thus, a “combined” embodiment of the invention is directed to a drug or therapeutic agent wherein, for example, a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody is a naked antibody and does not operably adhere to the antibody. Including when used in combination with In such cases, the agent or therapeutic agent may be used in an untargeted or targeted form. In "non-targeted form", the drug (especially a therapeutic agent) is generally used according to its standard use in the art. In a "targeted form", the agent is generally operably attached to a different antibody or targeted region that delivers the agent or therapeutic agent to the site of the angiogenic disease or tumor. The use of such targeted forms of biological agents (both diagnostic and therapeutic) is also fairly standard in the art.

In another “combined” embodiment of the invention, the VEGFR2 blocking anti-VEG
An F antibody or 2C3-based antibody is an immunoconjugate, wherein the antibody itself is operably associated with or binds to a drug or therapeutic agent. In certain preferred examples, the agent (including diagnostic and therapeutic agents) is a "2C3 targeted agent"
It is. Operable bonding includes all forms of direct and indirect bonding, as described herein and known in the art.

Use “in combination” (especially VEGFR2 blocking anti-VE in combination with a therapeutic agent)
GF antibodies or 2C3-based antibodies) also include combined compositions, medicaments, cocktails, kits, methods, and first and second medical uses, wherein the therapeutic agent comprises In the form of a prodrug. In such embodiments, the activating component that can convert the prodrug into a functional form of the drug is
It may again be operatively associated with a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody of the invention.

In certain preferred embodiments, the therapeutic compositions, combinations, medicaments, cocktails, kits, methods, and the first and second medical uses are “2C3
Prodrug combination ". As will be appreciated by those skilled in the art, the term "2
"C3 prodrug combination" means, unless otherwise specified, that the 2C3-based antibody is operably attached to a moiety capable of converting the prodrug to an active drug, wherein the 2C3-based antibody is attached to the prodrug itself. Does not mean to do. However, there is no requirement that the prodrug embodiments of the invention be used as a 2C3 prodrug combination. Thus, prodrugs can be used in any of the ways used in the art, including ADEPT and other forms.

Thus, the combined compositions, medicaments, cocktails, kits, methods and first and second medical uses are preferably described in terms of diagnostics and more preferably therapeutics. The combination includes a naked antibody and an immunoconjugate, a VEGFR2-blocking anti-VEGF antibody (eg, a 2C3-based antibody), wherein the practice of the in vivo embodiment of the present invention comprises the naked antibody or the immunoconjugate. Including pre-administration, co-administration or subsequent administration of conjugates and biological agents, diagnostic or therapeutic agents; some forms of antibodies, as far as some conjugated or unconjugated forms, And the overall supply of some form of biological agent, diagnostic or therapeutic agent is achieved.

[0189] Particularly preferred combined compositions, methods and uses of the present invention are directed to VEGFR2
Including blocking anti-VEGF antibodies and endostatin (US Pat.
No. 54,205, especially incorporated herein by reference). These are VE
Including the case where the GFR2 blocking anti-VEGF antibody or 2C3 construct is a naked antibody or immunoconjugate: and in the case of an immunoconjugate, where the VEGFR2 blocking anti-VE
The GF antibody or 2C3 binds endostatin (optionally with angiotensin); wherein the combined treatment method or use is pre-administration, simultaneous administration of endostatin (optionally with angiotensin). Administration or subsequent administration; as long as some conjugated or unconjugated forms include 2C3,
An overall supply of endostatin and optionally angiotensin is achieved.
VEGFR2 blocking anti-VEGF antibody or 2 operably associated with collagenase
C3-based antibodies are also provided as collagenases and, when delivered specifically to tumors, produce endostatin in situ and achieve similar benefits.

The above and other descriptions of the effects of the present invention on tumors are provided for simplicity and describe the combined mode of operation, types of adhesion factors, and the like. This descriptive approach should not be construed as any conservative expression or simplification of the advantageous properties of the VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies of the present invention. Thus, it is understood that such antibodies themselves will have anti-angiogenic and VEGF neutralizing properties (eg, neutralize the survival function of VEGF), and that immunoconjugates of such antibodies will have these properties. It is understood that these properties are combined with those of the adhesion factor; and, furthermore, the combined effects of the antibody and any adhesion factors are typically increased and / or expanded It is understood that.

Thus, the present invention provides compositions, pharmaceutical compositions, therapeutic kits and medical cocktails, optionally in at least a first composition or container. An effective amount of at least a first VEGFR2 blocking anti-VEGF antibody, optionally comprising one that binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA1595), or antigen binding of such an anti-VEGF antibody Fragments or immunoconjugates; and a biologically effective amount of at least a second biological agent, component or system.

"At least a second biological agent, component or system" is often a therapeutic or diagnostic agent, component or system, but often not a therapeutic or diagnostic agent, component or system. For example, at least the second biological agent, component or system can include a component for modifying the antibody and / or a component for attachment of another agent to the antibody. Certain preferred second biological agents, components or systems are prodrugs,
Or components for making and using prodrugs, including components for making the prodrug itself and components for adapting the antibodies of the present invention, such prodrugs or ADEPT implementations. Works in form.

When a therapeutic or diagnostic agent is included as at least a second biological agent, component or system, such therapeutic and / or diagnostic methods typically are associated with an angiogenic disease It is intended for use. Such factors are described in U.S. Pat.
Nos. 712,291, 5,753,230, 5,972,922, 5,639,757, WO98 / 45331 and WO98 / 16551 (
Each is an agent suitable for use in treating or diagnosing a disease or disorder, as disclosed in any one of the specific examples herein incorporated by reference.

If the disease to be treated is cancer, “at least a second anti-cancer agent” is included in the therapeutic kit or cocktail. The term “at least a second anticancer factor” refers to VE
Selected with reference to a GFR2 blocking anti-VEGF antibody or 2C3 construct, which is the first anti-cancer factor. Thus, the antibodies of the invention can be combined with chemotherapeutic agents, radiotherapeutic agents, cytokines, anti-angiogenic factors, apoptosis-inducing factors or anti-cancer immunotoxins or coag ligands.

[0195] As used herein, "chemotherapeutic agent" refers to a classic chemotherapeutic agent or drug used in the treatment of a malignancy. The term is used for simplicity, despite the fact that other compounds may be described professionally as chemotherapeutic agents in that they exert anti-cancer effects. However, "chemotherapy"
It has a different meaning in the art and is used according to its standard meaning. Many exemplary chemotherapeutic agents are described herein. One of skill in the art readily understands the use of chemotherapeutic agents and the appropriate dosage,
It can be reduced when used in combination with the present invention.

A new class of drugs, which may also be called “chemotherapeutic agents”, are factors that induce apoptosis. Any one or more of such drugs, including genes, vectors, antisense constructs and ribozymes, can also be used in combination with the present invention, as appropriate. Currently preferred second factors are anti-angiogenic factors (eg, angiostatin, endostatin (end)
ostatin), vasculostatin, canstatin and maspin).

Other exemplary anti-cancer factors include, for example, neomycin, podophyllotoxin, TNF-α, α _v β ₃ antagonist, calcium ionophore, calcium flux inducer, and any derivatives or prodrugs thereof. No.
Presently preferred anti-tubulin drugs include colchicine, taxol, vinblastine, vincristine, vindescine, combretastatin or a derivative or prodrug thereof.

An anti-cancer immunotoxin or coag ligand is further suitable as an anti-cancer factor.
An "anti-cancer immunotoxin or coag ligand" or targeting agent / therapeutic agent construct binds a targeting agent (a targetable or accessible component of a tumor cell, tumor vasculature or tumor stroma, and a therapeutic agent (cell (Including antibodies or antigen-binding fragments thereof) that operably adhere to damaging agents (including immunotoxins) and clotting factors (coag ligands). The “targetable or accessible component” of a tumor cell, tumor vasculature or tumor stroma is preferably a surface-expressed, surface-accessible, or surface-localized component, but a necrotic cell, or so. Components that would otherwise be released from damaging tumor cells or vascular endothelial cells (including cytosolic and / or nuclear tumor cell antigens) can also be targeted.

[0199] Both antibody and non-antibody targeting agents can be used, including: growth factors such as VEGF and FGF; tripeptide R that specifically binds to tumor vasculature. -Peptides containing GD; and other targeting components such as annexins and related ligands.

The anti-tumor cell immunotoxin or coag ligand is B3 (ATCC HB
10573), 260F9 (ATCC HB 8488), D612 (ATCC
HB 9796) and KS1 / 4. This KS1 / 4 antibody was prepared using the vector pGKC2310 (NRR
LB-18356) or the vector pG2A52 (NRRL B-18357).
).

Antitumor cell targeting agents that include an antibody or antigen-binding region thereof that binds to an intracellular component released from necrotic tumor cells are also contemplated. Preferably, such antibodies are present in cells that can be induced to become permeable, or in substantially all necrotic and normal cell ghosts, but outside normal mammalian living cells. A monoclonal antibody, or antigen-binding fragment thereof, that binds to an insoluble intracellular antigen that is absent or inaccessible.

US Pat. Nos. 5,019,368, 4,861,581, and 5
882,626 (each issued to Alan Epstein and co-workers) further describe methods for making and using antibodies specific for intracellular antigens that are accessible from malignant cells in vivo. And for the purpose of teaching, each is specifically incorporated herein by reference. The antibodies described are sufficiently specific for the inner cellular component of mammalian malignant cells, but not for the outer cellular component. Exemplary targets include histones,
All intracellular components that are specifically released from necrotic tumor cells are included.

Due to the process of tumor remodeling that occurs in vivo and causes necrosis of at least a portion of the malignant cells upon administration to an animal or patient having the vascularized tumor, the vascularized tumor is naturally a necrotic tumor cell. By virtue of the fact that such antibodies contain, such antibodies are localized to malignant cells. Furthermore, the use of such antibodies in combination with other therapies that enhance tumor necrosis is provided to enhance the efficacy of targeted and subsequent treatments.

Thus, these types of antibodies can be used to bind directly or indirectly to angiopoietin, as disclosed herein generally, and to vascularized It can be used to administer angiopoietin to necrotic malignant cells in living tumors.

US Pat. Nos. 5,019,368, 4,861,581, and 5
No., 882,626 (each of which is incorporated herein by reference), these antibodies may be used in combination diagnostic methods (see below) and the efficacy of anti-tumor treatment. It can be used in a method for measuring. Such methods generally involve the preparation and administration of a labeled version of the antibody, and measuring the binding of the labeled antibody to an internal cellular component target that is preferentially bound within necrotic tissue. . Thereby, the method images necrotic tissue. Here, the localized antibody concentration is indicative of the presence of a tumor and indicates a ghost of cells killed by anti-tumor treatment.

Anti-tumor stroma immunotoxins or coaguligands generally bind antibodies that bind to a connective tissue component, a basement membrane component, or an activated platelet component, as exemplified by binding to fibrin, RIBS or LIBS. Including.

The immunotoxin or coag ligand of the anti-tumor vasculature may be a ligand that binds to a surface-expressing, surface-accessible, or surface-localizing component of a blood-transporting vessel (preferably an intratumoral vessel) of a vascularized tumor. , Antibodies or fragments thereof. Such antibodies include those that bind to surface expressed components of intratumoral blood vessels of vascularized tumors. This includes: Intratumoral vasculature cell surface receptors (eg, endoglin (TEC-4 and TEC-11 antibodies))
, TGFβ receptor, E-selectin, P-selectin, VCAM-1, IC
AM-1, PSMA, VEGF / VPF receptor, FGF receptor, TIE
, Α _v β ₃ integrin, pleiotropin, endosialin and MHC class II proteins. The antibody may also bind to a cytokine- or coagulant-inducing component of intratumoral blood vessels. Certain preferred agents bind to an aminophospholipid (eg, phosphatidylserine or phosphatidylethanolamine).

Other anti-tumor vasculature immunotoxins or coaguligands may include antibodies or fragments thereof that bind to ligands or growth factors that bind to intratumoral vasculature cell surface receptors. Such antibodies are available from VEGF / VPF (GV3
9 and GV97 antibodies), FGF, TGFβ, ligands that bind to TIE, tumor-associated fibronectin isoforms, scatter factor / hepatocyte growth factor (HGF),
Includes antibodies that bind to platelet factor 4 (PF4), PDGF and TIMP. These antibodies or fragments thereof can also bind to the ligand: receptor complex or growth factor: receptor complex, but the ligand or growth factor or receptor is not in the ligand: receptor complex or growth factor: receptor complex. In that case, it cannot bind to ligand, growth factor or receptor.

Anti-tumor cells, anti-tumor stroma or anti-tumor vasculature antibody-therapeutic agent constructs comprise anti-angiogenic agents, such as plant-derived toxins, fungal-derived toxins, or bacterial-derived toxins; Tubulin drugs may include cytotoxic drugs. Lysine A chains and deglycosylated ricin A chains are often preferred. Anti-tumor cells, anti-tumor stroma or anti-tumor vasculature antibody-therapeutic agent constructs comprise a clotting factor or a second antibody binding region (coagulation factor) that binds a coagulant (a coagulation factor that acts directly or indirectly). To bind to). Tissue factor or tissue factor derivative (eg, shortened tissue factor)
An operable connection with is often preferred.

For the compositions, kits, and / or medicaments of the invention, the combined effective amount of the therapeutic agents may be contained in a single container or container means, or in different containers or container means. Is also good. The reaction mixtures are generally mixed together for combined use. Drugs formulated for intravenous administration are often preferred. An imaging component may also be included. The kit may also include instructions for use of at least the first antibody and one or more other included biological agents.

[0211] At least the second anticancer agent is VEGFR2-blocked (VEGFR2-b
locking), can be administered to an animal or patient substantially simultaneously with an anti-VEGF antibody or a 2C3-based therapeutic (eg, from a single pharmaceutical composition or two pharmaceutical agents administered together closely) From the composition).

Alternatively, at least a second anti-cancer agent can be administered to an animal or patient at a time sequential to the administration of a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic. “Sequential time”, as used herein, refers to “staggered (s
tagged), so that at least the second anticancer agent is VEG
Administered to the animal or patient at a different time from the administration of the FR2-blocking anti-VEGF antibody or 2C3-based therapeutic. Generally, two drugs are administered at a time interval effective to allow the two drugs to exert their individual therapeutic effect (ie, the two drugs are " Administered at biologically effective time intervals)). At least a second anticancer agent can be administered to an animal or patient at a biologically effective time prior to the VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic, or can be administered sequentially to those therapeutics. Alternatively, it can be administered to an animal or patient at a biologically effective time.

Thus, the present invention provides a method for treating an animal or patient having a vascularized tumor, comprising: (a) a method for substantially reducing tumor burden. Subjecting the animal or patient to one treatment; and (b) subsequently administering at least the first anti-angiogenic agent to the animal in an amount effective to inhibit metastasis from any remaining tumor cells. Or administering to the patient; wherein the first anti-angiogenic agent is at least a first VEGFR2-blocking anti-VEGF antibody or antigen-binding fragment thereof, or optionally, a monoclonal antibody Binds to substantially the same epitope as 2C3 (ATCC PTA 1595); optionally, the antibody or fragment is operable with a second anti-angiogenic agent. Sintering is the step.

Preferred first treatments include surgical resection and chemotherapeutic intervention.
Combined anti-angiogenic agents, such as angiopoietin 2 or a tumor-targeting angiopoietin 2 construct, may also be used.

Other methods of treatment for animals or patients with vascularized tumors include: (a) a first antibody-therapeutic agent construct in an amount effective to substantially induce tumor necrosis. Is administered to an animal or patient; wherein the first antibody-therapeutic agent construct comprises a surface-expressed component, a surface-accessible component, or a surface-accessible component of a tumor cell, tumor vasculature or tumor stroma. Including a therapeutic agent operably linked to the first antibody or antigen-binding fragment thereof that binds to the localization component; and (b) subsequently inhibiting metastasis from any remaining tumor cells. Administering a second antibody to the animal or patient in an effective amount; wherein the second antibody comprises at least a first VEGFR2-blocking anti-VEGF antibody or an antigen-binding fragment thereof. If it were a door, if necessary, monoclonal antibody 2C3 (A
TCC PTA 1595), which binds to substantially the same epitope as TCC PTA 1595);
And further optionally, the antibody or fragment is operably linked to a second anti-angiogenic agent.

In a particularly preferred embodiment, the invention provides VEGFR2-blocking anti-VEGF antibodies and 2C3-based antibodies for use in combination with prodrugs and ADEPT. In such compositions, combinations, medicaments, kits, methods and uses, the VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or fragment thereof may provide conversion or enzymological capabilities. Modified or operably linked (preferably covalently or conjugated) to at least a first converting agent or enzyme capable of converting at least one prodrug to an active form of the drug.

An antibody or fragment conjugated enzymatically or to an enzyme is combined with an initial separate formulation of a “prodrug”. A prodrug is an inactive or weakly active form of a drug that comprises a VEGFR2-blocking anti-VEGF antibody or 2
Upon contact with the enzymatic capacity, conversion function, or enzyme associated with the C3 antibody, it is converted to the active form of the drug.

Thus, preferably in a separate composition and / or container, provided is a kit comprising: (a) a biologically effective amount of at least a first VEGFR2-blocking anti-VEGF antibody or 2C3-based Antibodies, or fragments thereof, which have enzymatic function, preferably wherein the antibodies or fragments are operably linked or covalently linked to at least a first enzyme Or a conjugated, biologically effective amount of at least a first first VEGFR2-blocking anti-VE
A GF antibody or a 2C3-based antibody, or fragment thereof; and (b) a biologically effective amount of at least a first substantially inactive prodrug, wherein the VEGFR2-blocking anti-VEGF antibody or 2C3 antibody; The enzymatic function of the fragments, or a biologically effective amount of at least a first, that is converted to a substantially active drug by an enzyme conjugated, linked, or conjugated thereto. A substantially inert prodrug.

The present invention further provides advantageous methods and uses, including: (a) comprising at least a first VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody, or an antigen-binding fragment thereof. Administering a biologically effective amount of at least a first pharmaceutical composition to an animal or patient having a vascularized tumor, wherein the antibody or fragment has enzymatic function; Preferably, here, the antibody or fragment is operably linked, covalently linked, or conjugated to at least a first enzyme;
The antibody or fragment localizes to the vasculature, intratumoral vasculature, or stroma of the vascularized tumor after administration; and (b) subsequently, after a period of time, Administering to an animal or patient a biologically effective amount of at least a second pharmaceutical composition comprising an effective amount of at least one substantially inactive prodrug; wherein the prodrug is Is the enzymatic function of, or linked to, a VEGFR2-blocking anti-VEGF or 2C3 antibody or fragment localized within the vasculature, intratumoral vasculature, or stroma of this vascularized tumor Converted to a substantially active drug by an enzyme that has been, conjugated, or conjugated.

In certain other embodiments, the antibodies and immunoconjugates of the invention can be combined with one or more diagnostic agents, typically for use in connection with an angiogenic disease. . Accordingly, a range of diagnostic compositions, kits, and methods are encompassed by the present invention.

For the diagnosis and treatment of cancer, the diagnostic and imaging compositions, kits and methods of the present invention include in vivo and in vitro diagnostics. For example, a vascularized tumor is a diagnostically effective amount of a tumor diagnostic component, which is a tumor cell, tumor vasculature, or tumor stroma operably linked to a diagnostic imaging agent in vivo. Including at least a first binding region that binds to an accessible component of
It can be imaged.

Tumor imaging is preferably performed using a diagnostic component that includes at least a first binding region that binds to an accessible component of the tumor vasculature or tumor stroma and the vascularized tumor stroma and And / or implemented to provide an image of the vasculature. Any suitable binding region or antibody (eg, those described above for therapeutic constructs) can be used. The use of a detectably labeled VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody construct offers certain advantages, where the image formed is predicted at the binding site of the therapeutic agent used. It is a target.

A detectably labeled in vivo tumor diagnostic (whether or not a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody) may include: an X-ray detectable compound ( For example, bismuth (III), gold (III), lanthanum (III), or lead (II)); radioactive ions (eg, copper ⁶⁷ , gallium ⁶⁷ , gallium ⁶⁸ , indium ¹¹¹ , indium ¹¹³ , iodine ¹²³ , iodine ¹²⁵ , Iodine ¹³¹ , mercury ¹⁹⁷ , mercury ²⁰³ , rhenium ¹⁸⁶ , rhenium ¹⁸⁸ , rubidium ⁹⁷ , rubidium ¹⁰³ , technetium ^99m , or yttrium ⁹⁰ ); nuclear magnetic resonance isotopes (eg, cobalt (II), copper (II), chromium ( III), dysprosium (III), erbium (III), gadolinium (III), holmium (
III), iron (II), iron (III), manganese (II), neodymium (III)
, Nickel (II), samarium (III), terbium (III), vanadium (II), or ytterbium (III)); or rhodamine or fluorescein.

Pre-imaging prior to tumor treatment may be performed by: (a) at least first binding to an accessible component of tumor cells, tumor vasculature (preferred) or tumor stroma (preferred) Diagnostic agent operably linked to the binding region of
Administering to the animal or patient a diagnostically effective amount of a pharmaceutical composition comprising a VEGFR2-blocking anti-VEGF antibody or a diagnostic agent operably linked to a 2C3-based antibody construct); and (b) Subsequently, a detectably labeled first binding region (ie, a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody) bound to tumor cells, tumor blood vessels (preferred) or tumor stroma (preferred) is added. Detecting; thereby obtaining an image of the tumor, tumor vasculature and / or tumor stroma.

The treatment of cancer can also be performed by: (a) a tumor binding agent, or a VEGFR2-blocking anti-VEGF antibody or 2C3
A diagnostically minimal amount of at least a first detectably labeled tumor binding agent (preferably a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody comprising a diagnostic agent operably linked to a base antibody). Construct) is administered to an animal or patient having a vascularized tumor, thereby forming a detectable image of the tumor, tumor vasculature (preferred), or tumor stroma (preferred). Imaging the tumor; and (b) subsequently, administering a therapeutically optimal amount of at least a first naked VEGFR2-blocking anti-VEGF antibody or 2C3 antibody or therapeutic-antibody construct to such an antibody. Using and administering to the same animal or patient, thereby producing an anti-tumor effect.

Thus, provided are imaging and treatment formulations or medicaments, which generally comprise
Includes: (a) a diagnostically effective amount of a detectably labeled tumor binding agent (preferably V
EGFR2-blocking anti-VEGF antibody or 2C3-based antibody construct).
A first pharmaceutical composition comprising a detectable agent operably linked to a tumor binding agent or a VEGFR2-blocking, anti-VEGF antibody or 2C3-based antibody; and b) A second pharmaceutical composition comprising a therapeutically effective amount of at least one naked VEGFR2-blocking anti-VEGF antibody or 2C3 antibody, or a therapeutic-antibody construct using such an antibody.

The present invention also provides an in vitro diagnostic kit comprising at least one first composition or pharmaceutical composition comprising a biologically effective amount of at least one diagnostic agent, wherein the diagnostic agent comprises at least one The first VFGFR2 blocking anti-VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or an antigen binding fragment thereof, is operably linked.

The present invention still further provides a combined kit, wherein the diagnostic agent is intended for use in connection with a test performed outside of its body, preferably on a biological sample obtained from an animal or patient. provide. As such, the invention generally includes, in at least two different containers, at least a first composition, pharmaceutical composition, or medicinal cocktail, which comprises at least a first amount of a biologically effective amount. Blocking VEGFR2, an anti-VEGF antibody, optionally binding to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595), or an antigen-binding fragment or immunoconjugate of such an anti-VEGF antibody; and in vitro For use of a biologically effective amount of at least one diagnostic agent, component or system), a kit is provided.

A “diagnostic agent, component or system for use in vitro” is any diagnostic agent or combination of agents that allows for the diagnosis of one or more diseases having an angiogenic component. Thus, in vitro diagnostics include those suitable for use in the generation of diagnostic or prognostic information related to a disease or disorder as disclosed in any one of the following: US Patent No. 5,712. No. 291, No. 5,753, 23
No. 5, No. 5,972,922, No. 5,639,757, WO 98 /
45331 and WO 98/16551, each of which is hereby incorporated by reference in detail.

[0230] In terms of cancer diagnosis and treatment, the in vitro diagnostic agent binds to at least a first component that binds to an accessible component of tumor cells, tumor vasculature (preferably) or tumor stroma (preferably). It preferably comprises a diagnostic component comprising a binding region, which is operably linked to a "detectable or reporter factor" that is directly or indirectly detectable by an in vitro diagnostic test. A "detectable agent or reporter agent" that is directly detectable in vitro includes agents such as radiolabels and reporter agents that are detectable by immunofluorescence.

A “detectable or reporter factor” that is indirectly detectable in vitro is a factor that works with additional endogenous factors (eg, a detectable enzyme that contacts a chromogenic substrate to produce a colored product). including. Indirect detection in vitro also
It covers detectable components or reporter components or systems. These can be used in combination with at least one detection antibody having immunospecificity in the first binding region to produce tumor cells,
It comprises a first binding region that binds to an accessible component of the tumor vasculature (preferably) or tumor stroma (preferably). A “detection antibody” is preferably a “secondary antibody” that binds directly or indirectly to a detectable agent (eg, a radiolabel or an enzyme). Alternatively, a "secondary and tertiary antibody detection system" may be used, which system is combined with a secondary detection antibody having immunospecificity for the primary detection antibody to provide the immunospecificity for the first binding region. Wherein the secondary detection antibody is directly or indirectly bound to the detectable agent.

Description of Exemplary Embodiments Solid tumors and carcinomas account for over 90% of all cancers in humans.
Although the use of monoclonal antibodies and immunotoxins has been explored in the treatment of lymphomas and leukemias, these agents have been unexpectedly ineffective in clinical trials against carcinomas and other solid tumors (Abram and Oldham,
1985). The main reason that antibody-based treatment is ineffective is that macromolecules are not easily transported to solid tumors. Once inside the tumor mass,
These molecules cannot be uniformly distributed due to tight connections between tumor cells, fibrous stroma, interstitial pressure gradients, and the presence of binding site barriers (Dvorak et al., 1991).
).

In the development of new strategies for treating solid tumors, methods involving the targeting of tumor blood vessels, rather than tumor cells, offer distinct advantages. Effective destruction or blocking of tumor blood vessels arrests blood flow through the tumor and results in a surge of tumor cell death. Antibody-toxin and antibody-agglutinin constructs have already been used effectively for specific targeting and destruction of tumor vessels, resulting in tumor necrosis (Burrows et al.,
1992; Burrows and Thorpe, 1993; WO 93/1771.
5; WO96 / 01653; U.S. Patent Nos. 5,855,866; 5,877.
No. 5,965,132; No. 6,051,230; No. 6,
004,555; U.S. patent application Ser. No. 08 / 482,369 (paid fee paid October 20, 1998), each incorporated herein by reference).

When antibodies, growth factors or other binding ligands are used to specifically deliver coagulants to tumor blood vessels, such agents are referred to as “coagul ligands”.
igands) ". Currently, the preferred coagulants for use in coagulating ligands are truncated tissue factors.
(TTF) (Huang et al., 1997; WO 96/01653; US Pat. No. 5,877,289). TF is a major initiator of blood coagulation (initia
tor: initiator) (Ruf et al., 1991). At the site of injury, Factor VII / VIIa in the blood contacts and binds to TF on cells in perivascular tissue. The TF: VIIa complex activates Factor IX and Factor X in the presence of a phospholipid surface. This in turn leads to the formation of thrombin and fibrin, and ultimately to the formation of blood clots (Ruf and Edging
ton, 1994).

A range of suitable target molecules available on the tumor endothelium (but largely lacking on normal endothelium) has been described. For example, expressed targets may be utilized, such as: endoglin, E-selectin, P-selectin, VCAM-
1, a ligand reactive with ICAM-1, PSMA, TIE, LAM-1, VEG
F / VPF receptor, FGF receptor, α _V β ₃ integrin, preytropin or endosialin (US Pat. Nos. 5,855,866 and 5,877,2)
Nos. 89 and 6,004,555; Burrows et al., 1992; Bur.
rows and Thorpe, 1993; Huang et al., 1997; each of which is incorporated herein by reference).

Other targets that can be induced by the natural tumor environment or by the following human interventions are also targetable entities. This is described in U.S. Patent Nos. 5,776,427 and 6,036,955, each of which is incorporated herein by reference. When used in combination with prior suppression and tumor vasculogenesis in normal tissues, MHC class II antigens can also be used as targets (US Pat. Nos. 5,776,427; 6,004,554 and No.6,036,9
No. 55; each of which is incorporated herein by reference).

The adsorbed targets were VEGF, FGF, TGFβ, HGF, PF4, PDGF
, TIMPs, ligands that bind TIE, or another suitable group such as tumor-associated fibronectin isoforms (US Pat. No. 5,877,289).
No. 5,965,132; each of which is incorporated herein by reference). Fibronectin isoforms are ligands that bind to the integrin family of receptors. Tumor-associated fibronectin isoforms are targetable components of both tumor blood vessels and tumor stroma.

One currently preferred marker for such clinically targeted applications is receptor-associated VEGF. In fact, the assembly of VEGF: receptor complex is one of the most specific markers of tumor vasculature observed to date (US Pat. Nos. 5,877,289; 5,965,132). No. and No. 6,051,
No. 30, Lin-Ke et al., 1996; Dvorak et al., 1991b).

The VEGF: receptor complex presents an attractive target for specific delivery of drugs or other effectors to tumor endothelium. --- Because the tumor is
Enriched with cytokines and growth factors, and VEGF receptors are up-regulated under the hypoxic conditions found in most solid tumors (Mazure et al., 1996; Forsythe et al., 1996)
Waltenberger et al., 1996; Gerber et al., 1997; Kre.
mer et al., 1997). Upregulation of both the ligand and its receptor, specific for the tumor microenvironment, leads to receptors occupied at high concentrations in tumor vascular endothelium as compared to epithelium in normal tissues (US Pat. No. 5,877, 2
Nos. 89 and 5,965,132). Dvorak and allies also
A rabbit polyclonal antibody directed against the N-terminus of EGF was
(Bearing) Selectively stain tumor blood vessels after injection into syngeneic tumor (Lin-K
e et al., 1996).

The role of VEGF as a target for clinical intervention is not limited to immunotoxin or coaguligand treatment. In fact, VEGF is one of the key factors involved in solid tumor angiogenesis (Ferrara, 1995; Potgens et al., 1).
995), a strong permeability factor (Senger et al., 1983; Senger et al., 1).
990; Senger et al., 1986) and endothelial cell mitogens (Keck et al., 1989; Connolly et al., 1989; Thomas, 1996). The link between VEGF and angiogenesis has led to the proposal of various therapeutic strategies aimed at VEGF intervention (Siemeister et al., 1998).

A. VEGF and VEGF Receptor Vascular endothelial growth factor (VEGF) is a multifunctional cytokine induced by hypoxia and tumor mutations. VEGF is an early stimulator of the development and maintenance of a vascular network during embryonic development. It is a potent permeability inducer, endothelial cell chemoattractant, endothelial survival factor, and endothelial cell growth factor (Thoma
s. 1996; Neufeld et al., 1999). This activity is required for normal embryonic development (Fong et al., 1995; Shalaby et al., 1995). Because
Because targeted disruption of one or both alleles of VEGF results in embryonic lethality (Carmelieet et al., 1996; Ferrara et al., 199).
6).

VEGF is a key factor driving angiogenesis or angiogenesis in a number of physiological and pathological processes, and wound healing (Frank et al., 1995;
Burke et al., 1995), diabetic retinopathy (Alon, 1995; Malec).
aze, 1994), psoriasis (Detmar et al., 1994), atherosclerosis (Inoue et al., 1998), rheumatoid arthritis (Harada et al., 199).
8; Nagashima et al., 1999), solid tumor growth (Plate et al., 199).
4; Claffey et al. 1996).

A wide variety of cells and tissues produce VEGF, which is divided into at least five isoforms (121, 145, 165, 189, and 206 amino acids), splice variants encoded by the same gene. Exists (Hou
ck et al., 1991; Ferrara et al., 1991; Tischer et al., 1991.
). Two smaller isoforms (121 and 165) are secreted from the cells (Hook et al., 1991; Anthony et al., 1994). The secreted VEGF is a 38-46k monomer linked by two disulfide bonds.
It is an obligate dimer with Da.

The VEGF dimer has two well-characterized receptors (V
EGFR1 (FLT-1) and VEGFR2 (KDR / Flk-1)) and these receptors are selectively expressed on endothelial cells (Flt-1 and Flt-1).
lk-1 is a mouse homolog). The K _d for VEGF binding to VEGFR1 and VEGFR2 was 15-100 pM and 400-800 pM, respectively.
M (Terman 1994). A recently identified third cell surface protein (neuropilin-1) also binds VEGF with high affinity (Olander et al., 1991; DeVries et al., 19).
92; Terman et al., 1992; Soker et al., 1998).

VEGFR1 and VEGFR2 are type III receptor tyrosine kinases (
RTK III) family, which is characterized by seven extracellular IgG-like repeats, a single spanning transmembrane domain, and an intracellular split tyrosine kinase domain (Mustonen and Alitalo). , 1995). Until very recently, V
EGFR1 and VEGFR2 were thought to be almost exclusively expressed in endothelial cells (Mustonen and Alitalo, 1995).
. Although VEGFR1 and VEGFR2 have been reported to have different functions with respect to stimulating endothelial cell proliferation, migration and differentiation (Waltenberg)
ger et al., 1994; Guo et al., 1995), the precise role each receptor plays in VEGF biology and endothelial cell homeostasis was not clearly defined prior to the present invention.

Recent studies with knockout mice have shown VEGF, VEGFR1 and V
Each of the EGFR2 has been shown to be essential for angiogenesis, angiogenesis and embryonic development (Fong et al., 1995; Shalaby et al., 1995; Hiratsuk).
a et al., 1998). In lethal knockout studies, the phenotype associated with each receptor deficiency was different. Targeted disruption of VEGFR2 results in embryos that are defective in endothelial cell differentiation and are unable to form yolk sac blood glucose or terminate angiogenesis (Shalaby et al., 1995). VEGFR1 null mutants show impaired angiogenesis, disturbed aggregation of endothelial cells, and dilation of blood vessels (
Fong et al., 1995; Hiratsuka et al., 1998). VEGFR1 is
Clearly it has a vital biological role.

VEGFR1 has lower tyrosine kinase activity, but VEGFR1 has a higher affinity for VEGF than VEGFR2. This is VEG
The extracellular domain of FR1 is suggested to be particularly important. This hypothesis was strongly suggested by results from studies in knockout mice in which the tyrosine kinase domain of VEGFR1 was deleted while leaving the intact VEGF binding domain (Hiratsuka et al., 1998). VEGFR1-tyrosine kinase deficient embryos developed normal blood vessels and survived for a limited period of time (Hira
tsuka et al., 1998).

In addition to early knockout (Fong et al., 1995; Shalaby et al., 1995), studies by Hiratsuka et al. (1998) indicate that VEGFR1 has a significant biological role. However, the tyrosine kinase signal does not appear to be a critical factor. Macrophages from VEGFR1 knockout mice do not show VEGF-induced chemotaxis (Hiratsuka et al., 1998; incorporated herein by reference), thereby mediating this important biological response to VEGF. Interestingly, it has been shown that VEGFR1 is involved as a receptor involved in E. coli.

One group reported VEGFR2, a dominant signal receptor in VEGF-induced mitosis and permeability (Waltenberger et al., 1).
994; Zachary, 1998; Korpelainen and Alita.
lo, 1998). The function in macrophage migration and chemotaxis is similar to that of H
Although demonstrated in the work of iratsuka et al. (1998), the role of VEGFR1 in endothelial cell function is largely unclear.

[0250] Clauss et al. (1996; incorporated herein by reference) also report that V
EGFR1 was reported to have an important role in monocyte activity and chemotaxis. Indeed, cells of the macrophage / monocyte lineage express only VEGFR1, and this receptor is a receptor involved in mediating monocyte recruitment and procoagulant activity (Clauss et al., 1996). VEGF binding to VEGFR1 in monocytes and macrophages also acts by inducing intracellular calcium and inducing tyrosine phosphorylation (Clauss et al., 1).
996).

[0251] Binding of the VEGF dimer to the VEGF receptor is believed to induce receptor dimerization. Then, the dimerization of the receptor,
Specific tyrosine residues (Y80 inside the cells of FR2 and VEGFR1
1 and Y1175, and Y1213 and Y1333). This leads to a signal transduction cascade, which involves the activity of phospholipase Cγ (PLCγ) and phosphatidylinositol 3-kinase (PI3K) and increased intracellular calcium ions (Hood and Meininger, 1998; Hood et al., 1998). ;
Kroll and Waltenberger, 1998).

Although a number of groups have shown that nitric oxide (NO) is produced following VEGF activity of VEGFR2, the intracellular events further downstream in the VEGF-induced signal are less clear (Hood and Meininger). , 199
8; Hood et al., 1998; Kroll and Waltenberger, 19
98). The activity of VEGFR2, but not VEGFR1, caused VEGF to increase Src
And have been shown to activate the Ras-MAP kinase cascade (including MAP kinases, ERK1 and ERK2) (Waltenberge).
et al., 1994, 1996; Kroll and Waltenberger, 19
97).

In particular, the role of VEGFR1 in endothelial cell function is less clear, as Flt-1 tyrosine kinase deficient mice are viable and develop normal ducts (Hiratsuka et al., 1998). VEGF in endothelium
The primary biological role of R1 is to use non-signal ligand binding molecules, or VEGFR2
Has been suggested to play a role as a "decoy" receptor, which may be required to present VEGF to.

The association between VEGF and pathological angiogenic conditions has prompted various attempts to block VEGF activity. These include the generation of specific neutralizing antibodies to VEGF (Kim et al., 1992; Presta et al., 1997; Siussat et al., 1993; Kondo et al., 1993; Asano et al., 1995). Antibodies to the VEGF receptor have also been described (eg, US Pat. No. 5,840).
, 301 and 5,874,542, and, following the present invention,
WO 99/40118). US Patent No. 5,840,301
No. 5,874,542 is actually VEGF, not VEGF itself.
It has been suggested that blocking the receptor is advantageous for various reasons.

The soluble receptor constructs (Kendall and Thomas, 1993; A
iello et al., 1995; Lin et al., 1998; Millauer et al., 1996.
), Tyrosine kinase inhibitors (Siemeister et al., 1998), antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptor have also been reported (Saleh et al., 1996;
Cheng et al., 1996; Ke et al., 1998; Parry et al., 1999; each of which is incorporated herein by reference).

(B. Anti-VEGF Antibodies) (B1. Range of Antibody Properties) Applications of various inhibition methods include blocking angiogenesis and / or inhibiting tumor growth by interfering with VEGF signaling. It has been shown to be at least somewhat effective. Indeed, monoclonal antibodies to VEGF have been shown to inhibit human tumor xenograft growth and ascites formation in mice (Kim et al., 1993; Asano et al., 1995; 1998).
Mesiano et al., 1998; Luo et al., 1998a; 1998b; Borg;
strom et al., 1996; 1998).

Antibody A4.6.1 shows that VEG against both VEGFR1 and VEGFR2
A high-affinity anti-VEGF antibody that can block F binding (Kim et al., 19
92; Wiesmann et al., 1997; Muller et al., 1998). A4.6
. Alanine scanning mutagenesis and X-ray crystallography of VEGF bound by one Fab fragment showed that the epitope of VEGF bound by A4.6.1 was centered around amino acids 89-94. This structural data is
4.6.1 demonstrate that VEGF completely inhibits VEGF binding to VEGFR2, but most likely due to steric hindrance, but inhibits VEGF binding to VEGFR1 (Muller et al., 1998; Keyt et al., 1996; each of which is incorporated herein by reference).

A4.6.1 is the most widely used neutralizing anti-VEGF antibody in the literature to date. This is due to the growth of various human tumors in mice and VE
It has been shown to inhibit GF-induced vascular permeability (Brem, 1998; Ba).
ca et al., 1997; Presta et al., 1997; Mordenti et al., 1999.
Borgstrom et al., 1999; Ryan et al., 1999; Lin et al., 199;
9; each specifically incorporated herein by reference). A4.6.1 also inhibits ascites formation in a well-characterized human ovarian cancer mouse model and tumor dissemination in a novel metastatic mouse model. A4.6.1 has recently been humanized by monovalent phage display technology and is currently in Phase I clinical trials as an anticancer agent (Brem, 1998; Baca et al., 1997; Pr.
esta et al., 1997; each of which is incorporated herein by reference).

Despite some success in the art with neutralizing antibodies to VEGF, we have discovered that new antibodies, particularly VEGFR1 (FTL-1) and / or VEGFR2 (KDR / Flk- It has been found that those having a more precisely defined mode of interaction with 1) are advantageous for various reasons. For example, the development of anti-VEGF antibodies that selectively block the interaction of VEGF with only one of the two VEGF receptors has resulted in VEGFR1 and VEGFR2
Allows for a more accurate analysis of the pathway activated by VEGF in cells expressing both.

We have found that antibodies with defined epitope specificity that blocked VEGF, which binds only one receptor (VEGFR2), also have clinical advantages.
Of course, it was thought that they could have, depending on the maintenance of these inhibitory effects in the in vivo environment. (1998) studies in knockout mice show that both VEGFR1 and VEGFR2 have important biological roles. Prior to the present invention, VEGF was produced by only one of the two receptors.
Practical opportunities for therapeutic investigations regarding inhibiting the effects mediated by are hampered by the deficiency of effective tailor-made inhibitory agents.

We first developed a range of new anti-VEGF antibodies with different epitope specificities and properties. Six groups of hybridomas are provided that secrete a VEGF: receptor (Flk-1) complex or a monoclonal antibody against VEGF itself. Five of the antibodies did not block VEGF binding to its receptor, but one blocked this interaction (group 2C3) and inhibited VEGF-mediated proliferation of endothelial cells.

The antibodies of the groups 3E7, GV39M, and 2C3, all of which selectively localize to tumors after intravenous injection into mice bearing human tumor xenografts,
It is currently preferred for use in targeting, imaging and treating the vasculature or connective tissue of solid tumors.

A monoclonal antibody of the invention that recognizes the VEGF: receptor complex selectively localizes to tumor endothelial cells after injection into mice bearing human tumor xenografts. Monoclonal antibodies of the 2C3 group are also significantly localized in the connective tissue around the tumor vasculature and also in the surrounding tumor vasculature.

Antibodies that recognize the N-terminus react with receptor-bound VEGF by ELISA. GV39M and 11B5, in contrast to non-receptor bound VEGF,
Shows high specificity for receptor-bound VEGF. Probably GV3 on the N-terminus
The epitopes recognized by 9M and 11B5 are structural and
Created when GF binds to its receptor. Both of these antibodies are Ig
The fact that they are M and therefore large in size may be important for their selectivity for these VEGF: receptor complexes.

Anti-N-terminal antibody did not inhibit VEGF-mediated endothelial cell proliferation.
This suggests that the N-terminus of VEGF is not involved in receptor interaction and that antibodies against the N-terminus of VEGF do not interfere with VEGF-mediated signaling.

In contrast, 2C3 inhibits VEGF-mediated proliferation of endothelial cells with an IC ₅₀ of 3 nM. ¹²⁵ I-VE using KDR-expressing endothelial cells (ABAE cells)
GF binding studies demonstrated that 2C3 blocked VEGF binding to KDR in a concentration-dependent manner. Thus, 2C3 may neutralize KDR (VEGFR2) -mediated VEGF activity in vitro by interfering with VEGF binding to its receptor.

Immunohistochemical analysis revealed that GV39M, 11B5, 3E7, and 7G3 reacted moderately to strongly with vascular endothelium when applied directly to vascular endothelial fragments. . GV39M shows the highest specificity for non-tumor non-cells, with much less staining of tumor cells or connective tissue. 11B5, 3E7, and 7
G3 preferentially stains endothelial cells when applied at low concentrations, but at higher concentrations stains tumor cells and connective tissue separately.

The staining patterns observed for 11B5, 3E7 and 7G3 are typical of the type of staining seen when using a polyclonal antibody against VEGF that has no preference for a particular conformation of VEGF (Lin -Ke et al., 199
6; Plate et al., 1994; Claffey et al., 1996). Selective staining of endothelium by GV39M suggests that it binds to the VEGF: receptor complex on these cells, and the endothelial cell location of the receptor, and GV39M
Binds selectively to VEGF: sFlk-1 in ELISA.

Similarly, the wider staining patterns of 3E7 and 7G3 indicate that they are free VEG
Consistent with the ability to recognize both F and receptor-bound VEGF. However, 1
1B5 was predicted to have a more restricted staining pattern for endothelium. This is because it strongly favors VEGF: Flk-1 in the capture ELISA (see Table 2). It is possible that 11B5 can recognize VEGF binding to stromal components, giving a broader pattern of reactivity in tumor fragments.

3E7 and GV39M are selectively localized in vivo to vascular endothelial cells of tumor tissue, while 2C3 is localized to the perivascular connective tissue of tumors in addition to endothelium. 24 hours after intravenous injection into tumor-bearing mice, 3E7 was not detectable in endothelium of any tissue other than tumor. On the other hand, GV39M also
Binds to endothelial cells or mesenteric cells in the glomeruli of the kidney. The reason for the reactivity of GV39M with mouse renal mesenteric cells is not clear. It is possible that antibodies bind to the VEGF: receptor complex on normal endothelial cells of the kidney (Ta
Kahashi et al., 1995). However, localization studies in guinea pigs with homotypic strain 10 tumors showed that GV39M localized to tumor vessels but not glomeruli or vessels in other normal tissues.

The ability of 3E7 and GV39M to specifically localize to tumor endothelium is probably a result of at least two factors. First, the VEGF: receptor complex is relatively abundant on tumor blood vessels. The hypoxic tumor microenvironment stimulates VEGF expression by tumor cells and VEGF receptor expression by endothelial cells. Second, tumor vessels are more permeable than normal vessels (Yua
n et al., 1996), which may allow antibodies to gain more access to the VEGF: receptor complex, which appears to be enriched on surfaces remote from the lumen of the vessel (Lin-Ke). Hong et al., 1995).

In a previous study (1996) by Lin-Ke and co-workers, a rabbit polyclonal antibody directed against the N-terminus of rat VEGF was identified as TA3 / St.
It was found to localize to tumor endothelial cells after injection into mice with mouse breast or MOT ovarian cancer. In contrast, a rabbit polyclonal antibody directed against whole VEGF protein (Ab-618) did not specifically localize to endothelial cells in these tumors or elsewhere in the tumor itself.

Based on these results, Lin-Ke et al. (1996) concluded that the N-terminus of VEGF has the ability to bind antibodies after VEGF associates with the capillary endothelium and that cell-free VEGF or non-endothelial cells It was concluded that the pool of VEGF bound to was not sufficient to enrich anti-VEGF antibodies directed to non-N-terminal epitopes (Lin- Ke et al., 1996). The present results with 3E7 and GV39M directed to the N-terminus of VEGF support these conclusions.

However, the present invention provides that the 2C3 group of antibodies, which are directed against a non-N-terminal epitope of VEGF, localizes to both the vasculature and perivascular connective tissue of solid tumors in mice. Are significantly more surprising than the study of Lin-Ke et al. (1996). The present invention proposes that a "pool" of VEGF resides in the tumor stroma and, indeed, allows for enrichment of 2C3 in tumor mass. Such targeting of tumor stroma was not predictable from previous published studies. We believe that VEGF can be used for heparin sulfate proteoglycan (HSP) in tumors.
Considering that G) can be bound, an understanding of the mechanism of action is not particularly necessary in practicing the invention.

An early conclusion of the present invention was that antibodies from the GV39M and 3E7 groups were selectively localized to tumor endothelial cells in mice, whereas antibodies from the 2C3 group were associated with tumor endothelial cells and tumor perivascular. Localization in connective tissue. Since the distribution of VEGF and its receptor is similar in mice and humans, these antibodies
It is considered to show a similar pattern of localization in cancer patients. Therefore,
GV39M and 3E7 are predicted for use in delivering therapeutic or diagnostic agents to tumor vasculature in humans, while antibodies in the 2C3 group target therapeutic or diagnostic agents to tumor vasculature and tumor connective tissue As a vehicle for

B2. VEGFR2-Blocking Anti-VEGF and Anti-2C3 Antibodies Further studies of the 2C3 group of antibodies have revealed even more surprising properties.
An effective composition and use of the present invention has resulted.

An important finding of the present invention, made using ELISAs, receptor binding assays and receptor activation assays, was that the 2C3 group of monoclonal antibodies
Selectively blocking the interaction of GF with VEGFR2 (KDR / FLK-1) but not VEGFR1 (FLT-1).
The 2C3 antibody inhibits VEGF-induced phosphorylation of VEGFR2 and blocks VEGF-induced permeability, indicating that VEGFR2
As a receptor responsible for the permeability induced by VEGF.
The 2C3 antibody also has potent anti-tumor activity and arrests the growth of various established solid human tumors in art-recognized animal models of human cancer.

These findings demonstrate that 2C3 is not useful in analyzing the pathways activated by VEGF in cells expressing both VEGFR1 and VEGFR2, and demonstrate that 2C3 is not useful in tumor growth and survival processes. It emphasizes the importance of VEGFR2 activity. More importantly, they provide a unique mode of therapeutic intervention, including macrophage chemotaxis, osteoclast and cartilage resorption cell function (VEGF).
Allows specific inhibition of VEGFR2-induced angiogenesis without concomitant inhibition of FR1 (mediated by FR1).

Thus, the finding that considers 2C3 is, for the first time, only binding to VEGFR2 and VE
Provide motivation and means for making and using anti-VEGF antibodies that inhibit VEGF that does not bind to GFR1. Such antibodies are referred to briefly as "VEGFR2-blocking anti-VEGF antibodies", exhibit advantages in the art, and provide numerous advantages (both in unconjugated or "naked" form). And conjugated, ie, associated, with other therapeutic agents).

In Vitro Binding Studies of the Invention (ELISA Using Purified Receptor Protein)
And utilizing co-sedimentation assays) demonstrated that 2C3 blocks VEGF binding to VEGFR2. Surprisingly, 2C3 did not inhibit VEGF binding to VEGFR1 in any of the assays. The binding ELISA was repeated in different configurations to confirm initial results. In each configuration, the results showed that 2C3 did not interfere with the VEGF: VEGFR1 interaction. As a control for these studies, monoclonal antibody 3E7 (VE
Using an antibody) for terminal - NH ₂ in GF. This is the VEGF VEGFR
Neither binding to 1 nor binding to VEGFR2 was prevented.

Accordingly, the 2C3 group of antibodies of the present invention comprises other blocking antibodies to VEGF (A4.
(Including 6.1). A4.6.1 anti-VEGF antibody blocks VEGF from binding to both VEGF receptors. Crystallographic and mutagenesis studies have shown that the binding epitopes for VEGFR2 and VEGFR1 are concentrated toward the two symmetric poles of the VEGF dimer (Wiesmann et al., 1997; Muller et al., 1997). )
. Binding determinants on VEGF that interact with the two receptors are partially overlapping and distributed on four different segments that span the dimer surface (Mull
er et al., 1998). Antibody A4.6.1 binds to a region of VEGF that is within the receptor binding region of both receptors (Muller et al., 1998). 2C3
Binds to a region located near the VEGFR2 binding site, but does not bind to the VEGFR1 binding site.

Studies on the effect of 2C3 on VEGF-induced phosphorylation of the receptor have been shown to block VEGF-induced phosphorylation of VEGFR2. This also corresponds to the data discussed above, and further strengthens its role in VEGF-induced proliferation.

As with results from other studies, consistent VEGF-induced phosphorylation of VEGFR1 could not be demonstrated (De Vries et al., 1992; Waltenberg).
er et al., 1994; Davis-Smyth et al., 1996; Landgren et al., 1998). Therefore, it was not possible to easily determine whether 2C3 inhibits VEGF7-induced phosphorylation of VEGFR1. The low ability of VEGF for VEGFR1 phosphorylation led to suggest that VEGFR1 could act as a decoy receptor that captures VEGF and amplifies VEGFR2-mediated signaling rather than signaling receptors on endothelial cells. Get (Hira
tsuka et al., 1998). However, tyrosine phosphorylation of VEGFR1 by VEGF binding was reported by Kupprion et al. (1998) using human microvascular endothelial cells (HMEC) and by Sawano et al. (1996) using NIH 3T3 cells overexpressing VEGFR1. ing. In addition, Walte
(1994) have shown that VEGF-induced VEGFR1 activation can occur as a result of using an in vitro kinase assay. VEGFR1
The effect of 2C3 on VEGF-induced phosphorylation, or lack thereof, could be determined using one of the aforementioned cell types or an in vitro kinase assay.

The ELISA and cell binding data of FIG. 2 of the present invention show that the 2C3 antibody was VE
4 shows that VEGF is not completely blocked from binding to cells expressing both GFR1 and VEGFR2. The fact that 2C3 does not block VEGF binding to VEGFR1 means that the 2C3 antibody is an effective tool in demonstrating the role of VEGFR1 in biology of endothelial cells and other cell types.

The functional consequences of selectivity in 2C3 blocking VEGF from activating its receptor were tested using the Miles permeability assay in guinea pigs. Both 2C3 and A4.6.1 inhibited VEGF-induced permeability when IgG was at least 10-fold molar excess over VEGF. 3E7
And the control antibody did not inhibit VEGF-induced permeability, even at a 1000-fold molar excess. These results indicate that VEGFR2 is involved in VEGF-induced permeability.

A novel form of VEGF-C and two virus-induced VEGF-E variants
This finding, according to recent reports that binds EGFR2 but not VEGFR1, still maintains the ability to enhance vascular permeability. (Joukov et al., 19
98; Ogawa et al., 1998; Meyer et al., 1999). Presumably, various forms of VEGF signal through VEGFR2, which causes NO production, which in turn causes an increase in vascular permeability (Hood and Gr).
angel, 1998; Hood et al., 1998; Kroll and Walter.
Nberger, 1998; Murohara et al., 1998; Kupprion.
1998; Sawano et al., 1996; Fujii et al., 1997; Pare.
nti et al., 1998). This is indirectly indicated by VEGFR2 involvement, as NO production was shown against the result of VEGFR2 activation. However, Couple
In contrast, there is also some evidence found a strong correlation between VEGF-induced increase in vascular permeability and VEGFR1 expression in vitro, as in r et al. (1997).

2C3 inhibited the growth of several different human tumor types in vivo. 2 per week
The effect of 100 μg of 2C3 given once was due to the subcutaneous NCI-H358
Identical in NSCLC and A673 rhabdomyosarcoma tumors, where about 1
Up to the bar of 50mm ³ size, limited the growth of the tumor efficiently. Similar responses were seen in other tumor nodules (eg, HT29 and LS174T (both human adenomas of the colon)).

The magnitude of tumor growth inhibition by 2C3 was reported by other investigators using different neutralizing anti-VEGF antibodies (Asano et al., 1998; Mesiano et al.,
1998). The monoclonal rat anti-mouse VEGFR2 antibody also strongly blocked the growth of malignant human keratinocytes in mice via an anti-angiogenic mechanism (Skobe et al., 1997). The efficacy of 2C3 (similar to what others have found with different anti-VEGF antibodies) further demonstrates the role of VEGF in tumor angiogenesis and tumor growth. However, 2C3 provides a safer therapeutic based on the specific inhibitory properties discussed herein.

To analyze the effect of inhibiting VEGF activity in a setting closer to the human state, mice with established tumors were treated with 2C3. In this setting, 2C3
Treatment was with two aggressive human tumors, A673 rhabdomyosarcoma and LS174T
The growth of colon adenoma tumors was significantly delayed. The 2C3 antibody is a mouse-bearing NCI-H
Caused significant tumor regression in 358 NSLCL tumors.

[0290] Tumors treated with 2C3 or A4.6.1 disappeared after approximately 10 weeks of treatment to 30% and 35%, respectively, of their original size. Action is past 1
In a study allowed to be extended to day 00, an even more significant regression was observed. This result indicates that VEGF provides more than a mitotic signal for tumor endothelium.

The observation of the fact that tumors recede rather than quiescent suggests that VEGF provides more than an angiogenic signal for tumor endothelium. Benjam
(1999) recently reported that tumors contained a large fraction of immature blood vessels that had established contact with peripheral endothelial cells, and that these blood vessels depended on VEGF for survival. It is possible that neutralization of VEGF causes these immature blood vessels to undergo apoptosis, thereby causing a reduction in the vascular network present within the tumor. It is also possible that the kinetic process of vascular remodeling gives rise to tumors, is involved in both angiogenesis and vascular regression, and neutralization of VEGF
Prevent angiogenesis leading to a net shift towards vascular retraction.

The finding (if not more) that 2C3 completely suppresses tumor growth, similar to A4.6.1, indicates a major role for VEGFR2 in tumor angiogenesis. Multi-stage angiogenesis requires endothelial cell chemotaxis, metalloproteinase production, invasion, proliferation and differentiation. VEGFR1 may have no role in these processes or binds VEGF, and signaling receptors, VE
It may assist in the process by presenting to GFR2.

The comparative figures for 2C3 and A4.6.1 in the treatment of tumors are highly relevant: 2C3 is slightly more effective than A4.6.1, but only binds VEGFR2, Does not bind to VEGFR1. Therefore, the current study is VE
Shows that VEGFR1 does not play a significant role in GF-mediated tumor angiogenesis, and further indicates that VEGFR1-specific inhibitors cannot affect tumor angiogenesis. These results also indicate that 2C3 is similar to A4.6.1.
Or it is more effective, while producing fewer side effects.

The ability of VEGF to specifically bind to VEGFR2 and block activation of VEFGR2 is of importance in two areas of clinical relevance. First, VEGFR1 (Flt-1) is used by macrophages and monocytes to express VEGF.
It is thought to play an important role in recruiting macrophages and monocytes in tumors in expressing R1 and responding chemotactically to VEGF via VEGFR1 signaling (Clauss et al., 1996; Hiratsuka).
Et al., 1998; Akuzawa et al., 2000). Upon activation of macrophages, transcription of the flt-1 gene is stimulated through induction of Egr-1. Egr
-1 is the overlapping Egr-1 / Sp in the human flt-1 promoter
Binding to one transcription factor binding site, the flt-1 gene is a direct target of Egr-1 and provides evidence that transcription factors are predominantly induced in macrophage differentiation (Akuzawa et al., 2000).

In order to maintain macrophage activation required to generate a rigid anti-tumor response, inhibition of VEGFR1 signaling should be avoided. The specific blockade of VEGFR1 provided by the present invention therefore offers important advantages over A4.6.1 in tumor therapy. This is because the infiltration of macrophages is not impaired, allowing these cells to remove tumor cell debris from necrotic cells and promote tumor shrinkage. Using VEGFR2 blockade, anti-V
EGF antibodies (eg, 2C3) also allow macrophages to infiltrate and contribute to the overall anti-tumor effect by having the effect of killing cells directly to tumor cells.

Indeed, the present invention uniquely provides factors that are advantageous for use in all forms of anti-angiogenic therapy. These factors are advantageous by their ability to block VEGF angiogenic activity, but do not inhibit other beneficial effects of VEGF mediated through VEGFR1, such as effects on immunity and bone cells. A second area of clinical significance relates to the ability of antibodies prepared according to the present invention to function in vivo without inhibiting the beneficial effects of osteoclasts and cartilage resorbing cells. This implies the use of existing VEGFR2 blocking anti-VEGF antibody therapy (including 2C3) and is not associated with bone and / or cartilage side effects.

In vivo studies have shown that VEGF couples to hypertrophic cartilage remodeling, bone formation and angiogenesis during endochondral bone formation, and that VEGF is essential for cartilage remodeling (Gerber et al., 1999; specifically incorporated herein by reference). Soluble VEGFR1 receptor chimeric protein (Fl
Inactivation of VEGF1 mediated VEGF signaling by administration of (t- (1-3) -IgG) reduces trabecular bone formation and hypertrophic chondrocyte area by reducing recruitment and / or differentiation of chondrocytes. Have been shown to impair growth (Gerb
er et al., 1999).

It has further been shown that VEGF can replace macrophage colony stimulating factor (M-CSF) in favor of osteoclast function in vivo (Niida et al., 19).
99; specifically incorporated herein by reference). In studies using osteopetrotic (op / op) mice with deletions in osteoclasts that mutate the M-CSF gene, injection of recombinant human M-CSF (rhM-CSF) results in recruitment of osteoclasts And enable survival. Recent studies have shown that a single injection of recombinant human VEGF similarly induces osteoclast recruitment in op / op mice (Niida et al., 1999).

[0299] Niida et al. (1999) reported that since osteoclasts dominantly express VEGFR1 and that the activity of recombinant human placental growth factor 1 on osteoclast recruitment is comparable to that of rhVEGF, (op / op) reported beneficial effects of VEGF signaling in mice are mediated through VEGF receptor 1 (VEGF-1). These authors found that after VEGF was inhibited (VEGFR1
Receptor chimera protein, using VEGFR1 / Fc), rhM-CS
Although F-induced osteoclasts die, it was further shown that such effects were suppressed by co-injection of rhM-CSF. Osteoclasts supported by rhM-CSF or endogenous VEGF did not show significant differences in activity in vivo (
Niida et al., 1999).

Mutant op / op mice undergoing age-related recovery of osteopetrosis are associated with an increase in osteoclast numbers. In the study of Niida et al. (1999), most osteoclasts disappeared after injection of anti-VEGF antibody and endogenously produced VEGF
Was responsible for the appearance of osteoclasts in mutant mice. In addition, rhVEGF replaced rhM-CSF in favor of osteoclast differentiation in vitro. These results demonstrate that M-CSF and VEGF have overlapping functions in support of osteoclast function, and that VEGF acts via the VEGFR-1 receptor (Niida et al., 1999).

Thus, the first of the 2C3, VEGFR2 blocking, anti-VEGF antibodies of the present invention is VE
GF does not block binding and activating VEGFR1, but
It can be concluded that EGF blocks binding and activating VEGFR2. The antitumor effect of such VEGFR2 inhibition is clearly demonstrated. These results indicate that VEGFR2 is a VEGF receptor that mediates permeability and highlights its role in tumor angiogenesis. Thus, the present invention further confirms VEGF inhibition as a therapy for the treatment of solid tumors. More importantly, the present invention relates to a new range of VEGFR2 blockade, anti-VEGF antibodies (eg, 2C3-based antibodies for therapeutic intervention), and safe and effective to inhibit angiogenesis, especially in tumors and other diseases To provide an effective pharmaceutical use.

The advantages of the present invention are not limited to the elimination of side effects. However, these are important features that have significant advantages, especially in the treatment of children and patients with bone disorders, and the antibodies of the invention have a number of other advantages.

For example, antibody conjugates based on VEGFR2 blocking anti-VEGF or 2C3 antibodies can be used to deliver therapeutic agents to the tumor environment. In fact, the 2C3 antibody
It is shown herein that upon administration in vivo, it binds to both tumor vasculature and tumor stroma and does not bind to vasculature or connective tissue in normal organs or tissues. Therapeutic constructs based on the antibodies of the present invention therefore have the advantage of combining two functions in one molecule: the anti-angiogenic and therapeutic agent properties of the antibody or fragment thereof are selected for attachment.

Since VEGFR2 is a key receptor on the endothelium, binding of blocking VEGF to VEGFR2 is important for the anti-angiogenic effect. In this context, VEGF
R1 is expressed on endothelium but is non-signaling or passive. Thus, the inability of the antibodies of the present invention to block VEGF binding to VEGFR1 has no consequence for anti-angiogenesis and their effectiveness as anti-tumor factors. Indeed, VEGF binding to VEGFR1 occurs with prior art blocking antibodies
The ability of the antibodies of the present invention to bind to VEGF, rather than to inhibit, and also not substantially interfere with the VEGF-VEGFR1 interaction, enhances the drug delivery properties of these new antibodies.

We have reported that blocking antibodies are tumor-localized VEGs that are not bound to receptors.
It was further appreciated that binding to F would be expected to function to deliver a therapeutic agent to the tumor environment. Specifically, the present inventors provide such antibodies,
It was understood that it binds VEGF in the tumor stroma and delivers the therapeutic agent there. This provides a reservoir of drug around the endothelium, causes cytotoxic or other destructive effects on vascular endothelial cells, and exerts an antitumor effect.

VEGF bound to stroma or connective tissue is not bound to VEGF receptors (ie, cell surface receptors) in the traditional sense. Rather, VEGF
It binds to one or more connective tissue components (including proteoglycans (eg, heparan sulfate proteoglycans)) through the basic region of VEGF. Since these sequences (and the exons that encode them) are not within the VEGF121 protein (and the underlying DNA), this isoform should not be present in stroma in significant amounts. VEGF in the tumor stroma is often referred to as "free", but is still localized within the tumor, and "free" basically means non-receptor binding.

We have identified VE binding to one receptor, but not both receptors.
It is further inferred that antibodies that block GF can deliver therapeutic agents to the tumor environment by binding to VEGF-bound receptors on the vasculature. This is one of the most advantages of the present invention. That is, the definition of antibody is VEG
Blocks VEGF binding to FR2, and thus inhibits angiogenic signals from VEGF, but does not block VEGF binding to VEGFR1. In addition to reducing systemic side effects by maintaining VEGF1 mediated VEGF signaling in other cell types and tissues, these antibodies localize to the VEGF-VEGFR1 complex on tumor vasculature. And directly there to deliver the therapeutic agent.

Both VEGFR1 and VEGFR2 are up-regulated on tumor endothelial cells as opposed to endothelial cells in normal tissues. VEGFR1 is highly expressed on tumor vascular endothelium, making the targeted aspects of the invention particularly effective. In fact, VEGFR
1 is "non-signaling" in the endothelium, but if not at higher levels, is expressed at least as high as VEGFR2. Factors underlying this decrease include upregulation of VEGFR1 in response to both hypoxia and VEGF,
VEGFR2, on the other hand, is only up-regulated in response to VEGF and is not affected by hypoxia.

Although the role of VEGFR1 on endothelium remains uncertain, VEGFR1 can act as a decoy receptor, “capture” VEGF, and pass ligand to the signaling receptor, VEGFR2. To achieve this, it is expected that the decoy receptor will have a higher affinity for VEGF than the signaling receptor (which is the case). In this regard, and possibly also due to enhanced expression levels, the VEFGR2 blocking, non-VEGFR1 blocking antibodies of the present invention are ideal delivery agents for the treatment of tumors. Therapeutic conjugates of these antibodies can simultaneously inhibit angiogenesis through VEGFR2 and disruption of existing vasculature by delivering a therapeutic agent to the VEGF-VEGFR1 receptor complex.

We are by no means limited to the above scientific inferences as an explanation of the beneficial anti-angiogenic and tumor localizing properties of the antibodies of the present invention. The utility of the present invention is self-evident and does not require that the underlying theory be implemented, but we consider alternative mechanisms by which VEGFR2 blockade, non-VEGF
It was thought that the FR1 blocking antibody could effectively and specifically localize to the tumor vasculature.

Such antibodies can bind to VEGF and are related to Npn-d1 on the cell surface or another uncharacterized VEGF binding protein, or heparan sulfate on the surface of inner non-cells It can bind to VEGF bound to proteoglycans. Antibody localization may also be enhanced by binding to another member of the VEGF family of proteins associated with blood vessels (ie, VEGF-B, VEGF-C, VEGF-D), but this is more possible. Poor.

Another advantageous property of a VEGFR2 blocking anti-VEGF or 2C3 antibody of the invention is that
These antibodies, mediated through VEGFR2, neutralize the survival signal or "protective effect" of VEGF. This property, in addition to making itself more effective antibodies, makes them particularly useful by combining them with other factors that are hindered by the survival function of VEGF.

For example, VEGF protects the endothelium from radiation therapy. Thus, both naked antibodies and immunoconjugates of the invention are ideal for use in combination with radiation therapy. Yet a further advantage is provided by the use of such antibodies attached to a radiotherapeutic agent. This type of construct has three advantages: (1) exerting an anti-angiogenic effect through the antibody moiety; (2) exerting a tumor vasculature-destroying effect through the delivery of a radiotherapeutic agent; And (3) preventing the typical survival signal of VEGF from offsetting the effects of the radiotherapeutic agent.

Other constructs with similar synergistic effects include VEGFR2 associated with anti-tubulin drugs or prodrugs, anti-apoptotic factors and other anti-angiogenic factors
Blocking anti-VEGF antibody. The effects of factors or agents that cause apoptosis are antagonized by VEGF. Thus, the present invention improves the efficacy of such factors by neutralizing VEGF. VEGF's survival signal also opposes endostatin, which limits this treatment. Thus, in use in combination with endostatin, the VEGF of the invention
R2 blocking anti-VEGF, or 2C3 antibodies, neutralize VEGF and amplify the antitumor effects of endostatin. 2C3 or other VEGFR2 blocking anti-VEGF
Antibodies can also be used to specifically deliver collagenase to tumors, where collagenase produces endostatin in situ and achieves similar advantages.

In all such enhanced or synergistic combinations, the antibody and other factors can be administered separately, or a second factor can be linked to the antibody for specific delivery. (Ie, targeted delivery to VEGFR1)
. In combination with endostatin, chemical conjugates or recombinant fusion proteins are preferred. Because they offset the short half-life of endostatin, the short half-life of endostatin is currently a limitation of potential endostatin therapy. Combinations with tissue plasminogen activator (tPA), or targeted forms of tissue plasminogen activator (tPA) can also be used.

[0316] Additional benefits of the treatments of the present invention include the ability to reduce interstitial pressure. As VEGF-mediated increased permeability contributes to interstitial pressure, reduced signaling through VEFR2 reduces both permeability and interstitial pressure. In turn, this reduces the barrier for the drug to traverse the entire tumor tissue, so that tumor cells far from the vasculature can be killed. Long-term treatment can also be achieved when the compositions of the present invention have negligible or lesser immunogenicity.

B3.2C3 Antibody CDR Sequences The term “variable” as used herein in connection with an antibody means a particular part of the variable domain. This domain differs extensively in sequence between antibodies. The term is used in the binding and specificity of each particular antibody to a particular antibody. However, the variability is not evenly distributed across the variable domains of the antibody. It is concentrated in three segments called "hypervariable regions". Hypervariable regions are in both the light and heavy chain variable regions.

[0318] The more highly conserved portion of the variable domain is called the framework region (FR). Each of the variable domains of the native heavy and light chains contains four FRs (FR1, FR2, FR3 and FR4, respectively), mostly adopts a β-sheet configuration, and is bound to three hypervariable regions. , Form connecting loops, and in some cases, form part of a β-sheet structure.

The hypervariable regions in each chain are held together in close proximity to the FRs, and the hypervariable regions from the other chains contribute to the formation of the antigen-binding site of the antibody (Kabat et al., 19
91, specifically incorporated herein by reference). The constant domains are not involved directly in binding an antigen to an antigen, but exhibit various effector functions, such as participation of the antigen in antigen-dependent cellular toxicity.

The term “hypervariable region” as used herein refers to the amino acid residues of an antibody and is responsible for antigen binding. The hypervariable region is referred to as a "complementarity determining region" or "C
DR ”(ie, residues 24-3 in the light chain variable domain).
4 (L1), 50-56 (L2) and 89-97 (L3) and 31-35 (H1), 50-56 (H2) and 95-102 (H
3); Kabat et al., 1991, which is specifically incorporated herein by reference).
And / or residues from the “hypervariable region loop” (ie, residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain).
) And 26-32 (H1), 53-55 (H2) and 96-101 (H3)) in the heavy chain variable domain. "Framework" or "FR" residues are more variable domain residues than the hypervariable region residues as herein defined.

The DNA and deduced amino acid sequences of the Vh and Vk chains of the 2C3 ScFv fragment are provided herein as SEQ ID NOs: 6, 7, 8 and 9. These sequences include the CDRs 1-3 of the variable regions of the heavy and light chains of the antibody.

As described herein (Chapter C3), for defining the structural and functional information about biological molecules, a corresponding range, or even improved, molecules can be produced. This applies a VEGFR2 blocking anti-VEGF antibody of the invention, exemplified by the 2C3 antibody. Antigen binding and other functional properties of the antigen must be preserved, but there is a very high degree of skill in the art to produce comparable and even improved antibodies (once the reference antibody has been produced) I do. Such technical expertise applies to the production of additional antibodies with respect to the sequences and information provided herein. The antibody has improved or other desirable characteristics.

For equivalent antibodies, certain amino acids can be substituted for other amino acids in the antibody constant domain framework region or antibody variable domain framework region without appreciable loss of interactive binding capacity. That such changes are made in the DNA sequence encoding the antibody portion, and that the changes are conservative in nature (section C3, codon information in Table A, and support for site-specific mutagenicity). See technical details). Naturally, the number of changes to be made is limited, but is known to those skilled in the art.

[0324] Another type of variant is an antibody that has improved biological properties relative to the parent antibody from which it is produced. Such variants (ie, second generation compounds) are typically substituted variants that include one or more substituted hypervariable region residues of the parent antibody. A convenient method for producing such substitutional variants is affinity maturation using phage display.

In affinity maturation using phage display, several hypervariable region sites (eg, 6-7 sites) are mutated to produce all possible amino acid substitutions at each site. The antibody variants produced in this way are characterized by the M
When fused with 13 gene III products, they are displayed from filamentous phage particles in a monovalent fashion. The phage-displayed variants are then screened for their biological activity (eg, binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenicity can be performed to identify hypervariable region residues that significantly contribute to antigen binding.

Alternatively, or additionally, the crystal structure of the antigen-antibody complex is intended to be delineated and analyzed to identify contacts between the antibody and VEGF. Such contact residues and neighboring residues are candidates for substitution. Once such variants are generated, a panel of variants is subjected to screening as described herein and has similar properties in one or more related assays, but different properties or superior properties. Antibodies that have even better properties are selected for further development.

Thus, a further aspect of the invention relates to VEGFR2 blocking antibodies (anti-VEGF antibodies)
Isolated DNA segments and recombinant vectors encoding the CDR regions of the heavy and light chains (eg, 2C3 heavy and light chains) and related to the production and use of recombinant host cells through the application of DNA technology And these are such CDRs
Express the region.

Thus, the present invention relates to a DNA segment that can be isolated from any animal, preferably human or mouse, which does not contain total genomic DNA and
R2 blocking antibodies (anti-VEGF antibodies) can express the CDR regions of the heavy and light chains (eg, 2C3 heavy and light chains). As used herein, the term "DNA segment" refers to a DNA molecule that has been isolated without the total genomic DNA of a particular species. The term "DNA segment" also includes DNA segments and smaller fragments of such segments, as well as recombinant vectors (including, for example, plasmids, cosmids, phages, viruses, and the like).

Similarly, a DNA segment comprising the coding segment or the isolated gene portion encoding the purified CDR regions of the VEGFR2 blocking antibody (anti-VEGF antibody) heavy and light chains (eg, 2C3 heavy and light chains) , DNA segments, which contain such coding sequences (in certain aspects, regulatory sequences) and are isolated substantially apart from other naturally occurring gene or protein coding sequences. In this aspect, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or peptide coding unit. This functional term includes native antibody coding sequences and smaller engineered sequences that express or are adapted to express the appropriate antigen binding protein, polypeptide or peptide.

“Isolated substantially apart from other coding sequences” means that the coding segment or isolated gene portion of interest forms an important part of the coding region of the DNA segment, and A segment is a large portion of a naturally occurring coding DNA (eg, a large chromosomal fragment or other functional gene or c).
DNA coding region). Of course, this refers to the DNA segment originally isolated, and does not exclude genes or coding regions that have been subsequently added to that segment by hand.

In certain embodiments, the invention relates to isolated coding segments or isolated gene portions, and VEGFR2 blocking antibodies (anti-VEGF antibodies) heavy and light chains (eg, 2C3 heavy and light chains). Chain), which comprises at least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90% And most preferably comprises at least about 95% of the amino acid sequence region or such amino acid sequence identical to the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9; wherein the CDR region comprises SEQ ID NO: 7 or SEQ ID NO: 9
At least substantially retains the biological properties of the CDR regions of the amino acid sequence of

As disclosed herein, these sequences may contain certain biologically functionally equivalent amino acids or “conservative substitutions”. Other sequences are functionally non-equivalent amino acids or deliberately engineered to improve the properties of the CDRs or CDR-containing antibodies as known to those of skill in the art and as further described herein. "
And non-conservative substitutions.

The amino acid and nucleic acid sequences can include additional residues, such as additional N- or C-terminal amino acids or 5 ′ or 3 ′ sequences, and the sequence can be any of the criteria described above, preferably involving protein expression. (Including maintaining or improving biological protein activity) as well as still conforming to the sequences of the invention. The addition of terminal sequences includes various non-coding sequences flanking either the 5 'or 3' portions of the coding and also control regions.

Thus, the nucleic acid segments of the present invention may be combined with other DNA sequences (eg, promoters, polyadenylation signals, additional restriction sites, multiple cloning sites, other coding segments, etc.), such that their The overall length can vary considerably. Thus, nucleic acid fragments of almost any length can be utilized,
It is expected that the overall length is preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.

Thus, recombinant vectors form a further aspect of the present invention. Particularly useful vectors are expected to be those in which the coding portion of the DNA segment is placed under the control of a promoter. Generally, although not exclusively, recombinant or heterologous promoters are utilized (ie, promoters are usually not associated with coding sequences in their natural environment). Such promoters include bacterial promoters, as long as the promoter efficiently directs the expression of the DNA segment, in the cell type, organism, or even animal selected for expression.
It can include viral, eukaryotic and mammalian promoters.

The use of promoter and cell type combinations for protein expression is known to those of skill in the art of molecular biology. The promoter used can be constitutive (or inducible) and can be used under appropriate conditions to direct high level expression of the introduced DNA segment, for example, on a large scale of the recombinant protein or peptide. It is advantageous in production.

[0337] Expression of the nucleic acid sequences of the present invention can be conveniently achieved by one or more of any of the standard techniques known to those of skill in the art and further described herein. For example, the following description of recombinant expression of fusion proteins applies equally well to antibodies and antibody fragments that are not operably associated at the nucleic acid level with another coding sequence. (B4. Polyclonal antibodies) Means for preparing and characterizing antibodies are well known in the art. (
For example, Antibodies: A Laboratory Manual, C
(Old Spring Harbor Laboratory, 1988; incorporated herein by reference). To prepare a polyclonal antiserum, an animal is immunized with an immunogenic VEGF composition and antisera is collected from the immunized animal. A wide variety of animal species can be used for the production of antisera. Typically, animals used for the production of anti-antisera are rabbits, mice, rats,
Hamster, guinea pig, or goat. Rabbits are a preferred choice for the production of polyclonal antibodies because of their relatively large blood volumes.

The amount of the VEGF immunogenic composition used in the production of polyclonal antibodies
It will vary depending on the immunogen as well as the nature of the animal used for immunization. The following various routes may be used to administer the VEGF immunogen of the invention; subcutaneous, intramuscular,
Intradermal, intravenous, intraperitoneal, and intrasplenic. Polyclonal antibody production can be monitored by sampling the blood of the immunized animal at various times after immunization. A second, booster injection may also be given. The process of boosting and titering is repeated until the appropriate titer is achieved. Once the desired titer level is obtained, the immunized animal can be bled and serum can be isolated and stored. Animals can also be used to produce monoclonal antibodies.

As is well known in the art, the immunogenicity of a particular composition can be enhanced by the use of a non-specific stimulator of an immune response known as an adjuvant. An exemplary adjuvant is complete Freund's adjuvant (killed Mycobac)
nonspecific stimulators of the immune response containing terium tuberculosis); incomplete Freund's adjuvant; and aluminum hydroxide adjuvant.

It may also be desirable to boost the host immune system, such as may be achieved by linking VEGF to a carrier or by coupling VEGF to a carrier. Exemplary carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Ovalbumin,
Other albumins such as mouse serum albumin or rabbit serum albumin can also be used as a carrier. As is also known in the art,
A given composition may vary in its immunogenicity. However, production of antibodies to VEGF is not significantly difficult.

B5. Monoclonal Antibodies Various methods for producing monoclonal antibodies (MAbs) are also now well known in the art. The most standard monoclonal antibody production techniques generally start along the same system as the techniques for preparing polyclonal antibodies (Antibodies: A Laboratory Manual, Col.
d Spring Harbor Laboratory, 1988; incorporated herein by reference). A polyclonal antibody response is initiated by immunizing an animal with the immunogenic VEGF composition and, at the time when the desired titer level is obtained, the immunized animal can be used to produce a MAb.

MAbs can be readily prepared through the use of well-known techniques, such as the technique illustrated in US Pat. No. 4,196,265, which is incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected VEGF immunogen composition. The immunizing composition is administered in a manner effective to stimulate the antibody-producing cells. Rodents such as mice and rats are preferred animals, but the use of rabbit, sheep, and frog cells is also possible. Although the use of rats may provide certain advantages (Goding, 1986, pp. 60-61; incorporated herein by reference), mice are preferred. And BAL
B / c mice are the most commonly used and are most preferred as they generally give higher rates of stable fusions.

Following immunization, somatic cells with the potential to produce VEGF, specifically B lymphocytes (B cells), are selected for use in MAb production protocols. These cells can be obtained from a biopsy spleen, tonsil, or lymph node, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred. The former is because spleen cells are a rich source of antibody-producing cells at the dividing plasmablast stage, while the latter is because peripheral blood is readily available. Often, a panel of animals is immunized, and the spleen of the animal with the highest antibody titer is removed, and spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically,
Spleens from immunized mice contains approximately 5 × 10 ⁷ ~2 × 10 ⁸ lymphocytes.

The anti-VEGF antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortalized myeloma cell, typically a cell of the same species as the immunized animal. Myeloma cell lines suitable for use in the hybridoma-producing fusion procedure are preferably non-antibody-producing, have high fusion efficiencies, and then have a specific selection medium that supports the growth of only the desired fusion cells (hybridomas). It has an enzyme deficiency that renders it unable to grow in.

As is known to those skilled in the art, any of a number of myeloma cells can be used (Goding, pages 65-66, 1986; Campbell, 75-83).
1984, each of which is incorporated herein by reference). For example, when the immunized animal is a mouse, P3-X63 / Ag8, X63-Ag8.653,
NSl / 1. Ag 41, Sp210-Ag14, FO, NSO / U, MPC
-11, MPC11-X45-GTG1.7, and S194 / 5XX0 Bu
1 can be used; for rats, R210. RCY3, Y3-Ag1.2.3
, IR983F, 4B210, or one of the mouse cell lines listed above.
One may be used; and U-266, GM1500-GRG2, LICR-LO
N-HMy2 and UC729-6 are all useful in connection with human cell fusion.

Methods for producing hybrids of antibody-producing spleen cells or antibody-producing lymph node cells and myeloma cells are generally performed in the presence of factors (chemical or electrical) that promote cell membrane fusion. Mixing the somatic cells with the myeloma cells in a ratio of 1: 1 which can vary from about 20: 1 to about 1: 1 respectively. Fusion methods using Sendai virus are described in Kohler and Milstein (197).
5; 1976; each of which is incorporated herein by reference) and polyethylene glycol (PEG) (eg, 37% (v / v) P
Fusion methods using EG) are described in Geffer et al. (1977; incorporated herein by reference). The use of electrically induced fusion methods is also suitable (Goding, pp. 71-74, 1986; incorporated herein by reference).

The fusion procedure usually produces viable hybrids with low frequency (about 1 × 10 ⁻⁶ to 1 × 10 ⁻⁸ ). But this does not pose a problem. This is because viable fusion hybrids are distinguished from parental, non-fused cells, especially non-fused myeloma cells, which usually divide indefinitely, by culturing in selective media. The selection medium is generally a medium containing an agent that blocks de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserine blocks only purine synthesis. If aminopterin or methotrexate is used, supplement the medium with hypoxanthine and thymidine as a source of nucleotides (HAT medium). If azaserine is used, the medium is supplemented with hypoxanthine.

[0348] A preferred selection medium is HAT. Only cells that can function the nucleotide salvage pathway can survive in HAT medium. Myeloma cells contain key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPR).
T), and they cannot survive. Although B cells can function this pathway, they have a limited life span in culture and generally die within about two weeks. Thus, the only cells that can survive in the selection medium are myeloma cells and their hybrids formed from B cells.

The culture provides a population of hybridomas from which a particular hybridoma is selected. Typically, selection of hybridomas involves culturing cells by single clone dilution in microtiter plates and then testing the supernatants of individual clones for the desired anti-VEGF reactivity (after about 2-3 weeks). ). The assay should be sensitive, simple, and rapid (eg,
Radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunobinding assay (dot immunobinding assay)
y) etc.).

The selected hybridomas are then serially diluted and cloned into individual VEGF antibody producing cell lines. This clone is then expanded indefinitely and MA
b. Cell lines can be utilized for MAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type used to provide the somatic and myeloma cells for the original fusion. The injected animal develops a tumor that secretes the specific monoclonal antibody produced by the fused cell hybrid. The body fluid (eg, serum or ascites) of the animal may then be punctured to provide the MAb at a high concentration. Individual cell lines can also be cultured in vitro. Here, MAbs are naturally secreted into the culture medium, from which MAbs can be easily obtained at high concentrations.

The MAbs produced by any means are generally prepared, for example, by filtration, centrifugation, and various chromatographic methods (eg, HPLC or affinity chromatography), all of which are well known to those skilled in the art. Is further purified using Each of these purification techniques involves fractionation to separate the desired antibody from other components of the mixture. Analytical methods particularly suitable for preparing antibodies include, for example, protein A-Sepharose chromatography and / or
Or protein G-Sepharose chromatography.

B6. Antibodies from phagemid libraries Recombinant technology has now enabled the preparation of antibodies with the desired specificity from recombinant genes encoding a range of antibodies (Van Dijk). 198
9; incorporated herein by reference). Certain recombinant techniques include the isolation of antibody genes by immunological screening of a combinatorial immunoglobulin phage expression library prepared from RNA isolated from the spleen of the immunized animal (Morrison et al., 1986; Winter). And Milstein
1991; each of which is incorporated herein by reference).

For such a method, a combinatorial immunoglobulin phagemid library is prepared from RNA isolated from the spleen of an immunized animal, and phagemids expressing the appropriate antibody are expressed in cells expressing the antigen and control cells. Selection is performed by panning using cells. The advantage of this approach over conventional hybridoma technology is that about 10 ⁴ times as many antibodies can be generated and screened in one go, and that new specificity is generated by the combination of heavy and light chains. is there. This further increases the proportion of suitable antibodies produced.

One method for producing large repertoires of diverse antibody molecules in bacteria utilizes bacteriophage λ as a vector (Huse et al., 1989;
Which is incorporated herein by reference). The generation of antibodies using the λ vector is D
It involves cloning the heavy and light chain populations of the NA sequence into separate starting vectors. The vectors are subsequently randomly combined to form a single vector that directs co-expression of the heavy and light chains to form an antibody fragment. The heavy and light chain DNA sequences are obtained by amplification of mRNA isolated from spleen cells (or their hybridomas) from animals immunized with the selected antigen, preferably by PCR ^™ or related amplification techniques. )can get. The heavy and light chain sequences are typically prepared using primers that incorporate restriction sites at the ends of the amplified DNA segment to facilitate cloning of the heavy and light chain segments into the starting vector. Amplified.

Another method for generating and screening large libraries of antibody binding sites (ie, paratopes) that are completely or partially synthetic is from filamentous phage (eg, M13, fl or fd). Use display vectors. These filamentous phage display vectors (also called "phagemids") yield large libraries of monoclonal antibodies with diverse and novel immunospecificities. This technology uses the filamentous phage coat protein membrane anchor domain as a means to ligate gene products and genes during the assembly phase of filamentous phage replication, which clones and expresses antibodies from combinatorial libraries (Kang et al.,
1991; Barbas et al., 1991; each of which is incorporated herein by reference).

This general technique for filamentous phage display is described in US Pat. No. 5,658.
727, incorporated herein by reference. In most general recognition, this method provides a system for co-cloning and screening for ligand-binding specificities preselected from an antibody gene repertoire using a single vector system. By screening the isolated members of the library for preselected ligand-binding ability, the expressed antibody can be isolated using any convenient means for isolating the gene encoding the member from the library. It is possible to correlate the binding ability of molecules.

[0357] Linking expression and screening can be used to target the fusion polypeptide to the periplasm of bacterial cells to allow for the assembly of functional antibodies, and to facilitate convenient screening of library members of interest. It is achieved in combination with targeting of the fusion protein to the coat of filamentous phage particles during phage assembly. Periplasmic targeting is provided by the presence of a secretory signal domain in the fusion polypeptide. Targeting to phage particles is achieved by binding the filamentous phage coat protein membrane anchor domain (ie, cpII) in the fusion polypeptide.
I or a membrane anchor domain from cpVIII).

The diversity of a filamentous phage-based combinatorial antibody library is determined by modifying one or more complementarity determining regions of the cloned heavy chain genes of the library by shuffling the heavy and light chain genes. , Or by introducing random mutations into the library by an error-prone polymerase chain reaction. Additional methods for screening phagemid libraries are described in US Pat. Nos. 5,580,717; 5,427,908; 5,403,484; and 5,223,4.
No. 09, each of which is incorporated herein by reference.

Alternative Methods for Screening Large Combinatorial Antibody Libraries
A method has been developed that incorporates filamentous bacteria such as M13, fl or fd.
Utilizes the expression of diverse populations of heavy and light chain sequences on the surface of a phage (US Patent
5,698,426; incorporated herein by reference). Various heavy chains (
The two populations of Hc) and light chain (Lc) sequences are ^TM ). These populations contain the necessary elements for expression
Cloned into a separate M13-based vector. Heavy chain vector
Gene VII such that translation of the sequence produces a gVIII-Hc fusion protein.
I (gVIII) coat protein sequence. The two vector populations are Hc
And only the vector portion containing the Lc sequence is ligated into a single circular vector.
As such, they are randomly combined.

The combined vector directs co-expression of both Hc and Lc sequences for assembly and surface expression of the two polypeptides at M13 (US Pat. No. 5,698,426; herein). Incorporated by reference in the book). The combining step involves combining different Hc code sequences and L
Randomly link c-code sequences. This vector sequence, provided from each independent vector, is required for the generation of live phage. Furthermore, pseudo gV
Since the III sequence is contained in only one of the two starting vectors, the Lc-associated g
Co-expression of a functional antibody fragment as a VIII-Hc fusion protein
It cannot be achieved on the phage surface until the vector sequence is ligated into a single vector.

[0361] Surface expression of the antibody library is performed in an Amber suppressor strain.
The amber stop codon between the Hc and gVIII sequences does not link the two components in the non-suppressor strain. Isolating the phage produced from the non-suppressor strain and infecting the suppressor strain links the Hc sequence to the gVIII sequence during expression. Cultivating the suppressor strain after infection can be performed with gVII
All antibody species in the library can be co-expressed on the surface of M13 phage as an I fusion protein (gVIII-Fab fusion protein). Alternatively, the DNA can be isolated from a non-suppressor strain and then introduced into the suppressor strain to achieve the same effect.

The surface expression library is screened for specific Fab fragments that bind the preselected molecule by standard affinity isolation procedures. Such methods include, for example, panning (Parmley and Smith,
1988; incorporated herein by reference), affinity chromatography and solid phase blotting procedures. Panning is preferred because high titer phage can be screened easily, quickly and in small volumes. In addition, this procedure may select for trace Fab fragment species within a population. This trace fragment species is otherwise undetectable and does not grow to a substantially homogeneous population. Selected Fab fragments can be characterized by sequencing the nucleic acid encoding the polypeptide after expansion of the phage population.

Another method for generating diverse libraries of antibodies and screening for desired binding specificities is described in US Pat. Nos. 5,667,988 and 5,757.
No. 9,817, each of which is incorporated herein by reference. This method uses degenerate oligonucleotide and primer extension reactions to incorporate degenerate CDR regions of the variable domains of the immunoglobulin variable heavy and light chains, and display of the mutagenized polypeptide on the surface of the phagemid. Preparation of a library of heterodimeric immunoglobulin molecules in the form of a phagemid library to be used. Thereafter, the display proteins are screened for the ability to bind to a preselected antigen.

Methods for producing heterodimeric immunoglobulin molecules generally comprise (1)
(2) introducing a randomized binding site into a phagemid display protein vector and a region homologous to the CDR of the antibody V region gene into a phagemid display vector; Displays that can express different putative binding sites, each displayed on a phagemid surface display protein, introduced by primer extension with an oligonucleotide containing a degenerate region to generate a randomized coding sequence. Forming large populations of vectors; (3) expressing display proteins and binding sites on the surface of filamentous phage particles; and (4) using affinity techniques (eg, panning of phage particles against a preselected antigen). Fur expressed on the surface The particles were isolated (screening), by this, comprises isolating one or more phagemid containing a display protein containing a binding site that binds a preselected antigen.

A further variation of this method for generating a diverse library of antibodies and screening for the desired binding specificity is described in US Pat.
No. 2,892, which is incorporated herein by reference. In this method, only the heavy chain sequence is used, which is randomized at every nucleotide position encoding either the CDRI hypervariable region or the CDRIII hypervariable region, and the genetic variable in the CDRs. Sex is generated independently of any biological process.

In this method, two libraries are manipulated to genetically shuffle oligonucleotide motifs within the backbone of the heavy chain gene structure. CDRI or CD
By any random mutation of RIII, the hypervariable region of the heavy chain gene was reconstructed to yield a highly diverse collection of sequences. The heavy chain protein encoded by the collection of mutated gene sequences required only one of the two immunoglobulin chains, but retained the potential to have all of the binding properties of the immunoglobulin.

In particular, the method is performed in the absence of an immunoglobulin light chain protein. A phage library displaying the modified heavy chain protein is incubated with the immobilized ligand, and a clone encoding a recombinant protein that specifically binds to the immobilized ligand is selected. The bound phage are then dissociated from the immobilized ligand and amplified by growth in bacterial host cells. Individual viral plaques, each expressing a different recombinant protein, are expanded, and individual clones can then be screened for binding activity.

B7. Antibodies derived from human lymphocytes In vitro immunization, or antigenic stimulation, can also be used to generate human anti-VEGF antibodies. Using such a technique, peripheral blood lymphocytes from a normal healthy subject can be stimulated simply by stimulating antibody-producing cells with VEGF in vitro. Such "in vitro immunization" generally involves antigen-specific activation of non-immune B lymphocytes in a mixed population of lymphocytes (mixed lymphocyte culture, MLC). In vitro immunization can also be supported by B cell growth and differentiation factors and lymphokines. Antibodies produced by these methods are often
IgM antibody (Borrebaeck et al., 1986; incorporated herein by reference).

Another method is described (US Pat. No. 5,681,729, incorporated herein by reference), wherein human lymphocytes producing mainly IgG (or IgA) antibodies are obtained. Can be This method transplants human lymphocytes into an immunodeficient animal in a general sense so that the human lymphocytes are "entrapped" in the animal's body;
Immunizing the animal with the desired antigen to produce human lymphocytes that produce antibodies specific to the antigen; and collecting the antibody-producing human lymphocytes from the animal. Accordingly, the human lymphocytes produced are used to immortalize human lymphocytes producing antibodies, the resulting immortalized human source lymphocytes producing antibodies are cloned, and from the cloned immortalized human source lymphocytes. Monoclonal antibodies can be produced by collecting monoclonal antibodies specific to the desired antigen.

[0370] Immunodeficient animals that can be used in this technique are immunodeficient animals that do not show rejection when the animals are transplanted with human lymphocytes. Such animals can be prepared artificially by physical, chemical, or biological treatment. Any immunodeficient animal can be used. Human lymphocytes can be obtained from human peripheral blood, spleen, lymph nodes, tonsils, and the like.

“Uptake” of transplanted human lymphocytes in an animal can be achieved by simply administering human lymphocytes to the animal. The route of administration is not limited and can be, for example, subcutaneous, intravenous, or intraperitoneal. The dose of human lymphocytes is not limited,
And usually it may be 10 ⁶ to 10 ⁸ lymphocytes per animal. The immunodeficient animal is then immunized with the desired VEGF antigen.

After immunization, human lymphocytes are collected from blood, spleen, lymph nodes or other lymph tissues by any conventional method. For example, mononuclear cells are Ficoll-H
Ypaque (specific gravity: 1.077) can be separated by centrifugation, and monocytes can be collected by plastic dish adsorption. Contaminating cells from immunodeficient animals can be removed by using an antiserum specific for the animal cells. Antisera can be obtained, for example, by immunizing a second different animal with spleen cells of an immunodeficient animal and collecting serum from the different immunized animal. Treatment with the antiserum can be performed at any stage. Human lymphocytes can also be recovered by immunological methods using human immunoglobulins expressed on the cell surface as markers.

By these methods, one or more selected VEGF epitope specific I
Human lymphocytes predominantly producing gG and IgA antibodies can be obtained. Then
Monoclonal antibodies are obtained from human lymphocytes by immortalization, selection, cell growth, and antibody production.

B8. Transgenic Mice Containing Human Antibody Libraries Recombinant technology is now available for the preparation of antibodies. In addition to the recombinant immunoglobulin phage expression library disclosed above, another molecular cloning approach is to prepare antibodies from transgenic mice containing a human antibody library. Such techniques are disclosed in US Pat. No. 5,545,807.
, Which is incorporated herein by reference.

In the most general sense, these methods can be rearranged in their germline to encode genetic material encoding at least a portion of an immunoglobulin of human origin, or a repertoire of immunoglobulins. Includes the production of transgenic animals into which the genetic material has been inserted. The inserted genetic material can be produced from human sources or can be produced synthetically. The material can encode at least a portion of a known immunoglobulin, or can be modified to encode at least a portion of a modified immunoglobulin.

The inserted genetic material is expressed in the transgenic animal to produce immunoglobulins derived at least in part from the inserted human immunoglobulin genetic material. The genetic material contains immunoglobulins that are partially derived from the inserted genetic material, even if the inserted genetic material is incorporated at the wrong position in the germline or at the wrong relative position. The repertoire is found to be rearranged in the transgenic animal so that it can be produced.

The inserted genetic material can be in the form of DNA cloned into a prokaryotic vector such as a plasmid and / or cosmid. Larger DNA
The fragment was prepared using a yeast artificial chromosome vector (Burke et al., 1987).
Or by introducing a chromosomal fragment (Richer and Lo, 1989; incorporated herein by reference). The inserted genetic material can be introduced into the host in a conventional manner (eg, by injection or other procedures into a fertilized egg or embryonic stem cell).

In a preferred aspect, a host that does not initially carry the genetic material encoding the immunoglobulin constant region, such that the resulting transgenic animal uses only inserted human genetic material when producing immunoglobulins. Animals are used. This ultimately creates a host in which the relevant genetic material has been removed, using a naturally occurring mutant host that lacks the relevant genetic material, or artificially, for example, in a cell line. To do so, either can be achieved by making variants.

When the host animal carries genetic material encoding an immunoglobulin constant region,
Transgenic animals carry naturally-occurring genetic material and inserted genetic material, and obtain immunoglobulins from naturally occurring genetic material, inserted genetic material, and mixtures of both types of genetic material. Produce. In this case, the desired immunoglobulin can be obtained by screening hybridomas from transgenic animals, for example, using the phenomenon of allelic exclusion of antibody gene expression or differential chromosomal loss.

[0380] Once a suitable transgenic animal has been prepared, the animal is simply immunized with the desired immunogen. Depending on the nature of the material to be inserted, the animal will produce a chimeric immunoglobulin of mixed mouse / human origin if the genetic material of foreign origin encodes only a portion of the immunoglobulin, for example. Alternatively, the animal may produce fully foreign immunoglobulins (eg, completely human) if the genetic material of foreign origin encodes whole immunoglobulins.

[0392] Polyclonal antisera can be produced from transgenic animals following immunization. Immunoglobulin producing cells can be removed from animals producing the immunoglobulin of interest. Preferably, monoclonal antibodies are produced from the transgenic animal, for example, by fusing spleen cells from that animal with myeloma cells and screening the resulting hybridomas to select those that produce the desired antibody. Is done. Suitable techniques for such a process are described herein.

In an alternative approach, the genetic material is added to the animal in a manner such that the desired antibody is produced in a body fluid, such as the animal's serum, or in an external secretion, such as milk, colostrum, or saliva. Can be incorporated. For example, the genetic material encoding at least a portion of a human immunoglobulin in vitro is inserted into a mammalian gene encoding a milk protein, and the gene is then introduced into a fertilized egg of the mammal, for example, by injection. By doing so, the egg can grow into an adult female mammal producing milk containing immunoglobulins derived at least in part from the inserted human immunoglobulin genetic material. The desired antibody can then be recovered from the milk. Suitable techniques for performing such a process are known to those skilled in the art.

The transgenic animals described above are usually of a single isotype, more particularly those that are essential for B cell maturation (eg, IgM and possibly IgD).
Of human antibodies. Another preferred method for producing human anti-VEGF antibodies is described in U.S. Patent Nos. 5,545,806; 5,569,82.
No. 5, No. 5,625,126; No. 5,633, 425; No. 5,611
No., 016; and 5,770, 429, each of which is incorporated herein by reference. There, transgenic animals are described that can convert from the isotype required for B cell development to another isotype.

In the development of B lymphocytes, the cells first produce IgM with a binding specificity determined by the productively rearranged V _H and V _L regions. As a result, each B cell and its progeny synthesize antibodies with the same L and H chain V regions, but they can exchange H chain isotypes. The use of the mu or delta constant region is largely determined by alternate splicing, with IgM and I
Allows gD to be co-expressed in a single cell. Other heavy chain isotypes (γ, α, and ε) are only expressed in nature after a gene rearrangement event has deleted the Cmu and Cdelta exons. This gene rearrangement process is called isotype switching, and typically occurs by recombination between so-called switch segments (excluding deltas) located immediately upstream of each heavy chain gene. Individual switch segments are between 2 kb and 10 kb in length and consist primarily of short, repeated sequences.

For these reasons, it is preferred that the transgene incorporates a transcriptional regulatory sequence within about 1-2 kb upstream of each switch region to be used for isotype switching. These transcription regulatory sequences preferably include promoter and enhancer elements, and more preferably 5 ′ flanking (ie, upstream) that naturally binds to the switch region (ie, occurs in a germline configuration).
Including the region. Although the 5 'flanking sequence from one switch region can be operably linked to a different switch region for transgene construction, in some embodiments,
Preferably, each switch region incorporated in the transgene construct has a 5 'flanking region immediately upstream in the naturally occurring germline configuration. Sequence information relating to immunoglobulin switch region sequences is known (Mills et al., 1990; Sideras et al., 1989; each incorporated herein by reference).

US Pat. Nos. 5,545,806; 5,569,825; 5,625.
No. 126, No. 5,633,425; No. 5,661,016;
In the method described in US Pat. No. 770,429, the human immunoglobulin transgene contained in the transgenic animal functions correctly through a B cell development pathway leading to isotype switching. Thus, in this method, these transgenes are constructed to produce isotype switching and one or more of the following: (1) high level and cell type specific expression; (2) functional gene rearrangement; (3
) Activation of allelic exclusion and response to allelic exclusion, (4) expression of a sufficient primary repertoire, (5) signaling, (6) somatic hypermutation, and (
7) Control of the transgene antibody locus during the immune response.

A key requirement for transgene function is the generation of a primary antibody repertoire that is sufficiently diverse to trigger a secondary immune response to a wide range of antigens.
The rearranged heavy chain gene chains are each encoded by several exons,
It consists of a tandem array of signal peptide exons, variable region exons and regions of the multi-domain constant region. Each of these constant region genes encodes the constant portion of a different class of immunoglobulin. During B cell development, the V region proximal constant region is deleted, leading to the expression of a new heavy chain class. For each heavy chain class, alternative patterns of RNA splicing give rise to both transmembrane and secreted immunoglobulins.

The human heavy chain locus is approximately 200 V gene segment spanning 2 Mb, approximately 4
Almost 30D gene segment spanning 0 kb, cluster within 3 kb 6
It consists of one J segment and nine constant region gene segments that span almost 300 kb. The full length locus spans approximately 2.5 Mb of the distal portion of the long arm of chromosome 14. All members of the six known _VH families, D
And J gene segments and heavy chain transgene fragments containing the μ, δ, γ3, γ1 and α1 constant regions are known (Berman et al., 1988; incorporated herein by reference). Genomic fragments containing all necessary gene segments and regulatory sequences from the human light chain locus are similarly constructed.

[0389] Expression of successfully rearranged immunoglobulin heavy and light chain transgenes usually has a dominant effect by suppressing the rearrangement of endogenous immunoglobulin genes in transgenic non-human animals. However, in certain embodiments, for example, transswitching between a transgene and an endogenous Ig sequence prevents the formation of a hybrid immunoglobulin chain comprising a human variable region and a non-human (eg, mouse) constant region. It is desired to perform complete inactivation of the endogenous Ig locus. Using embryonic stem cell technology and homologous recombination, the endogenous immunoglobulin repertoire can be easily eliminated. In addition, suppression of the endogenous Ig gene can be achieved using various techniques, such as antisense techniques.

In another aspect of the invention, it may be desirable to generate trans-switched immunoglobulins. Antibodies comprising such chimeric transswitch immunoglobulins can be used for a variety of applications in which it is desirable to have a non-human (eg, mouse) constant region, for example, to maintain effector function in a host cell. . The presence of a mouse constant region has the advantage of providing a mouse effector function (eg, ADCC, mouse complement fixation) over a human constant region, eg, such that such a chimeric antibody can be tested in a mouse disease model. Can be given. Subsequent to animal testing, human variable region coding sequences are isolated, for example, by PCR ^™ amplification or cDNA cloning from a source (hybridoma clone), and the desired sequence for encoding a human sequence antibody more suitable for human therapeutic use. Can be spliced into a sequence encoding a human constant region.

B9. Humanized Antibodies In general, human antibodies have at least three possible advantages for use in human therapy. First, since the effector moiety is human, human immune cells are used to destroy target cells more efficiently, for example, by complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC). Can interact better with other parts of the system. Second, the human immune system does not recognize antibodies as foreign. Third, the half-life in human circulation is similar to naturally occurring human antibodies, allowing smaller and smaller doses to be given.

[0392] Various methods for preparing human anti-VEGF antibodies are provided herein. In addition to human antibodies, "humanized" antibodies have many advantages. "Humanized" antibodies are generally chimeric or variant monoclonal antibodies from mouse, rat, hamster, rabbit or other species that retain human constant and / or variable region domains or particular alterations. Techniques for producing so-called "humanized" anti-VEGF antibodies are well known to those skilled in the art.

[0393] Humanized antibodies also share the preceding advantages. First, the effector moiety is still human. Second, the human immune system does not recognize the framework or constant region as foreign, and therefore the antibody response to such injected antibodies is less than for completely foreign mouse antibodies. Third, the injected humanized antibody
Contrary to the injected mouse antibody, it has a half-life more similar to the naturally occurring human antibody, and also gives smaller and less frequent doses.

[0391] Many methods for producing humanized antibodies have been described. The controlled rearrangement of antibody domains linked through protein disulfide bonds to form new artificial protein molecules or "chimeric" antibodies can be utilized (Koniecz
ny et al., 1981; incorporated herein by reference). Recombinant DNA technology can also be used to construct gene fusions between DNA sequences encoding mouse antibody variable light and heavy chain domains and human antibody light and heavy chain constant domains (Morrison et al., 1984). ; Incorporated herein by reference).

A DNA sequence encoding the antigen binding portion or complementarity determining region (CDR) of a mouse monoclonal antibody can be inserted by molecular means into a DNA sequence encoding the framework of the human antibody heavy and light chains (Riechmann) Et al., 1988
). The expressed recombinant product is termed the "new form" or humanized antibody, and comprises the human antibody light or heavy chain and C, the antigen-recognition portion of a mouse monoclonal antibody.
Includes DR framework.

Another method for producing humanized antibodies is described in US Pat. No. 5,639,641, which is incorporated herein by reference. This method provides, by reproduction, a humanized rodent antibody with improved therapeutic efficacy due to the presentation of a human surface in the variable region. In this method: (1) Generate a position alignment of the pool of antibody heavy and light chain variable regions and provide a set of heavy and light chain variable region framework surface exposure positions. Here, the alignment positions for all variable regions are at least about 98% identical; (2) the heavy and light chain variable region framework surface exposed sets of amino acid residues are identical to the rodent antibody (or fragment thereof). (3) a set of heavy and light chain variable region framework surface exposed amino acid residues is identified that is closest and identical to this set of rodent surface exposed amino acid residues; (4) The heavy and light chain variable region framework surface exposed amino acid residues defined in step (2) are within 5% of any atom of any residue in the complementarity determining region of the rodent antibody. (5) excluding the amino acid residues as in step (3), the set of heavy and light chain variable region framework surface exposed amino acid residues identified in step (3); Humanized rodent antibody having a slip specificity is produced.

A similar method for the production of humanized antibodies is described in US Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,533.
No. 0,101, each of which is incorporated herein by reference. These methods involve producing a humanized immunoglobulin having one or more complementarity determining regions (CDRs) and additional amino acids from a donor immunoglobulin and framework regions derived from an accepted humanized immunoglobulin. I do. Each humanized immunoglobulin chain typically comprises, in addition to the CDRs, CDRs, such as one or more amino acids immediately adjacent to the CDRs in the donor immunoglobulin or one or more amino acids within about 3% estimated by molecular modeling. And amino acids from the donor immunoglobulin framework that can interact with and result in binding affinity. Heavy and light chains are disclosed in U.S. Patent Nos. 5,693,762;
No. 5,585,089; and 5,530,101, each of which is incorporated herein by reference. , Any combination, or all, can be designed. When combined with an intact antibody, a humanized immunoglobulin is substantially non-immunogenic in humans and retains substantially the same affinity for the original antigen as the donor immunoglobulin.

[0398] Additional methods for producing humanized antibodies are described in US Patent No. 5,565,332.
And No. 5,733,743, each of which is incorporated herein by reference. This method combines the concept of humanizing antibodies with a phagemid library, which is also described in detail herein. In a general sense,
This method utilizes sequences derived from the antigen binding site of an antibody or population of antibodies to the antigen of interest. Thus, for a single rodent antibody, the sequence comprising a portion of the antibody's antigen binding site may be combined with a diverse repertoire of human antibody sequences that can be combined to create a complete antigen binding site.

The antigen binding site created by this process is created by CDR grafting in that only portions of the sequence of the original rodent antibody appear to contact the antigen in a similar manner. Is different from the antigen-binding site. The selected human sequences appear to differ in sequence, and make alternative contacts with antigens from those of the original binding site. However, the constraints imposed by the binding of portions of the native sequence to the antigen and the shape of the antigen and its antigen binding site appear to drive new contacts of the human sequence to the same region or epitope of the antigen. Therefore, this process is called "epitope imprint selection" (EIS).

Starting with animal antibodies, one process results in the selection of antibodies that are partially human antibodies. Such antibodies can be sufficiently similar in sequence to human antibodies to be used directly or after alteration of a few key residues in therapy. Sequence differences between the rodent components of selected antibodies having a human sequence may be due to, for example, site-directed mutagenesis of individual residues or CDR grafting of entire loops, Can be minimized by substituting different residues with However, antibodies with fully human sequences can also be made. Therefore, EI
S describes a method for making a partially human antibody that binds to the same epitope as an animal or partially human antibody, respectively, or a fully human antibody that binds to the same epitope as an animal or partially human antibody, respectively. provide. In the EIS, a repertoire of antibody fragments can be displayed on the surface of filamentous phage, and the gene encoding the fragment with antigen binding activity can be selected by binding of the phage to the antigen.

Additional methods for humanizing antibodies contemplated for use in the present invention are described in US Pat. Nos. 5,750,078; 5,502,167; 5,705,154.
No. 5,770,403; No. 5,698,417; No. 5,693.
Nos. 493; 5,558,864; 4,935,496; and 4,816,567, each of which is incorporated herein by reference. WO 98/45331 and WO 98/45332 are particularly useful,
And to further illustrate the principle of humanization when applied to anti-VEGF antibodies,
It is incorporated herein by reference.

B10. Mutagenesis by PCR ^™ Site-directed mutagenesis is a useful technique in the preparation of individual antibodies through specific mutagenesis of the underlying DNA. This technique further enhances the rapid ability to prepare and test sequence variants with the aid of one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA, whether or not to humanize. provide.

Although many methods are appropriate for use in mutagenesis, the use of the polymerase chain reaction (PCR ^™ ) is generally presently preferred. This technology has a certain DN
It provides a quick and efficient method for introducing a desired mutation into the A sequence. The text below specifically describes the use of PCR ^™ to introduce point mutations in a sequence, such as can be used to change the amino acids encoded by a given sequence. The adaptation of this method is also suitable for introducing restriction enzyme sites into DNA molecules.

In this method, a synthetic oligonucleotide is designed to incorporate a point mutation at one end of the amplified segment. After PCR ^TM, amplified fragments by treatment with Klenow fragment, is blunt-ended, then, the blunt-ended fragments are ligated into a vector to facilitate sequence analysis, and subcloned .

To prepare the DNA of the template desired to be mutagenized, the D
NA is subcloned into high copy number vectors (eg, pUC19) using restriction sites adjacent to the region to be mutated. The template DNA is then prepared using a miniprep of the plasmid. Based on the parent sequence but containing the desired point mutation and flanked at its 5 'end by a restriction enzyme site,
Suitable oligonucleotide primers are synthesized using an automated synthesizer.
Primers homologous to template DNA of about 15 bases are generally required. Primers can be purified by denaturing polyacrylamide gel electrophoresis, but this is not absolutely necessary for the use of PCR ^™ . The 5 'end of the oligonucleotide should then be phosphorylated.

The template DNA should be amplified by PCR ^™ using an oligonucleotide primer containing the desired point mutation. MgC in amplification buffer
The concentration of l ₂ is typically about 15 mM. Generally, about 20-25 cycles of PC
^RTM should be performed as follows: denaturation at 95 ° C for 35 seconds;
Hybridization for 2 min; and extension for 2 min at 72 ° C. PCR ^™ generally involves a last cycle extension of about 10 minutes at 72 ° C. After the last extension step, about 5 units of Klenow fragment should be added to the reaction mixture and incubated at about 30 ° C. for another 15 minutes. The exonuclease activity of the Klenow fragment is required to flatten the ends and make them suitable for blunt-end cloning.

The resulting reaction mixture should generally be analyzed by non-denaturing agarose or acrylamide gel electrophoresis to confirm that this amplification has yielded the putative product. The bulk of the mineral oil was then removed, extracted with chloroform to remove the remaining oil, extracted with buffered phenol,
The reaction mixture is worked up by concentration by precipitation with 0% ethanol. Next, approximately half of the amplified fragment should be digested with restriction enzymes that cut at the adjacent sequences used in the oligonucleotide. This digested fragment is purified on a low gelling / melting point agarose gel.

To subclone this fragment and check for point mutations, the two amplified fragments are subcloned into the appropriately digested vector by blunt end ligation. This is due to the fact that plasmid DNA can be subsequently prepared using minipreps. used to transform E. coli. The amplified portion of the plasmid DNA is then analyzed by DNA sequencing to confirm that the correct point mutation has been made. Taq DN
This is important because A polymerase introduces additional point mutations in the DNA fragment.

The introduction of point mutations can also be effected using a series of PCR ^™ steps. In this procedure, the two fragments containing the mutation are annealed to each other and extended by mutual-initiated synthesis. This fragment is then
Amplified by the PCR ^™ step, thereby avoiding the blunt end ligation required for the above protocol. In this method, the template DN
Preparation of A, preparation of oligonucleotide primers and first PCR ^™ amplification
This is performed as described above. However, in this process, the selected oligonucleotides should be homologous to the template DNA for a stretch of between about 15 and about 20 bases and must also overlap each other by no more than about 10 bases.

In a second PCR ^™ amplification, using the conditions described above, each amplified fragment and each adjacent sequence primer, and from about 20 to about 25
Run PCR ^™ for cycles between. Again, this fragment is subcloned and the point mutations are checked for accuracy using the steps outlined above.

It is generally preferred to introduce mutations by amplifying as small a fragment as possible using any of the methods described above. Of course, parameters (eg, the melting temperature of the oligonucleotide, which is generally affected by the GC content and the length of this oligo) should also be carefully considered. The performance of these methods, and, if necessary, their optimization, is known to those of skill in the art and various publications (eg, Current
Protocols in Molecular Biology, 1995)
Are described further.

When performing site-directed mutagenesis, Table A can be used as a reference.

[0413]

[Table 1]

B11. Antibody Fragments and Derivatives Independent of the source of the original VEGFR2 blocking anti-VEGF antibody, any one of an intact antibody, an antibody multimer, or various functional antigen binding regions of the antibody
One can be used in the present invention. An exemplary functional region is the s of an anti-VEGF antibody.
Includes cFv, Fv, Fab ', Fab and F (ab') ₂ fragments. Techniques for preparing such constructs are well known to those of skill in the art and are further exemplified herein.

[0415] The choice of antibody construct can be influenced by a variety of factors. For example, prolonged half-life can result from active reabsorption of intact antibodies in the kidney (a characteristic of Fc pieces of immunoglobulins). Therefore, IgG-based antibodies are expected to show slower blood clearance than their Fab 'counterparts. However, Fab ′ fragment-based compositions generally exhibit better tissue penetration capabilities.

[0416] Antibody fragments can be obtained by proteolysis of whole immunoglobulins by papain, a non-specific thiol protease. By papain digestion,
Two identical antigen binding fragments (the "Fab fragment" and the remaining "F
Fab fragments, each having a single antigen binding site).

[0417] Papain must first be activated by reducing the sulfhydryl group in the active site with cysteine, 2-mercaptoethanol or dithiothreitol. Heavy metals in the stock enzyme should be removed by chelation with EDTA (2 mM) to compensate for maximum enzyme activity. The enzyme and the substrate are usually mixed together in a weight ratio of 1: 100. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. The end of this digestion was monitored by SDS-PAGE, and the various fractions were
-Should be separated by Sepharose or ion exchange chromatography.

A common procedure for the preparation of F (ab ′) ₂ fragments from IgG of rabbit and human origin is limited proteolysis by the enzyme pepsin. These conditions (100 × antibody excess w / w pH 4.5 in acetate buffer, 37 ° C.) suggest that the antibody is cleaved C-terminal to the disulfide bond in the heavy chain. Mouse I
The rate of digestion of gG can vary from subclass to class, and conditions should be chosen to avoid significant amounts of fully degraded IgG. In particular, IgG _2b is susceptible to complete degradation. Other subclasses require different incubation conditions (all of which are known in the art) to produce optimal results.

Pepsin treatment of intact antibodies results in F (a) with two antigen binding sites
b ′) ₂ fragments are obtained, and may still crosslink the antigen. Digestion of rat IgG by pepsin, 0.1 M acetate buffer, pH 4.5, dialysis in, then require conditions including 4 hours incubation using 1% w / w pepsin; IgG ₁ and IgG _2a Digestion is improved when first dialyzed against 0.1 M formate buffer, pH 2.8 at 4 ° C. for 16 hours, followed by acetate buffer. IgG _2b was prepared from staphylo in 0.1 M phosphate buffer, pH 7.8.
Incubation for 4 hours at 37 ° C. in coccus V8 protease (3% w / w) produces more consistent results.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab 'fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. F (ab ') ₂ antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are known.

An “Fv” fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region is a tight covalent bond (con-cova).
It is composed of a dimer of one heavy chain variable domain and one light chain variable domain. It is within this configuration that the three hypervariable regions of each variable domain interact to define an antigen binding site on the surface of the _VH - _VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has a lower affinity than the entire binding site, but the ability to recognize and bind the antigen Having.

“Single-chain Fv” or “sFv” antibody fragments comprise the V _H and V _L domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, Fv polypeptides further comprise a polypeptide linker between the _VH and _VL domains, which allows the sFv to form the desired structure for antigen binding.

The following patents and patent applications disclose the present teachings regarding the preparation and use of functional antigen-binding regions of antibodies, including scFv, Fv, Fab ′, Fab and F (ab ′) ₂ fragments of anti-VEGF antibodies. No. 5,855,866; US Pat. No. 5,965,132; US Pat. No. 6,051,230 for the purpose of still further supplementing US Pat. No. 6,004,555; and No. 5,877,289; and US patent application Ser. No. 08 / 482,3.
No. 69 (Payment fee was paid on October 20, 1998). WO98 /
45331 also describes the variable, hypervariable, and complementarity determining regions (CD
R) ( ) Are incorporated herein by reference for purposes including still further describing and teaching the preparation of

“Diabody” is a small antibody fragment having two antigen-binding sites, which fragment is a heavy chain connected to the light chain variable domain (V _L ) in the same polypeptide chain (V _H -V _L ). A chain variable domain (V _H ). 2 on the same chain
By using a linker that is too short to allow pairing between the two domains, these domains are forced to pair with the complementary domain of another chain and create two antigen binding sites I do. The diabody is EP404,0
97 and WO 93/11161, each of which is specifically incorporated herein by reference. “Linear antibodies” (which can be bispecific or monospecific) are described in Zapata et al. (1995) (
As described are specifically incorporated) by reference herein, a pair of tandem Fd segments which form a pair of antigen binding regions _{(V H -C H 1-V} H -C H 1
)including.

In using Fab ′ fragments or antigen-binding fragments of an antibody, additional advantages may be derived by modifying the fragment to increase its half-life, with the attendant benefits of tissue penetration. A variety of techniques can be used, such as manipulation or modification of the antibody molecule itself, and also coupling to an inert carrier. Any binding should be carefully approached for the sole purpose of increasing the half-life, rather than delivering the drug to the target, where Fab 'and other fragments penetrate tissue To be selected. Nevertheless, attachment to non-protein polymers such as PEG is contemplated.

Thus, modification rather than binding is based on modifying the structure of the antibody fragment so as to make the antibody fragment more stable and / or reduce the rate of catabolic effects in the body. One mechanism for such modification is the use of D-amino acids instead of L-amino acids. The introduction of such modifications requires that subsequent rigorous testing of the resulting molecule be necessary to ensure that the resulting molecule still retains the desired biological properties. Traders understand. Additional stabilizing modifications include the use of the addition of a stabilizing moiety, either at the N-terminus or C-terminus, or both, which is generally used to increase the half-life of a biological molecule. Is done. For illustrative purposes only, it may be desirable to modify the termini by acylation or amination.

A modest binding modification for use with the present invention involves incorporating a salvage receptor binding epitope into the antibody fragment. Techniques for accomplishing this include mutation of the appropriate region of the antibody fragment, or incorporation of the epitope as a peptide tag attached to the antibody fragment. WO9
No. 6/32478 is specifically incorporated herein by reference for the purpose of further describing such techniques. A salvage receptor binding epitope is typically a region of three or more amino acids from one or two laps of an Fc domain that is transferred to a similar position in an antibody fragment. WO98 / 4
The 5331 salvage receptor binding epitope is hereby incorporated by reference for use with the present invention.

B12. Binding and Functional Assays The present invention has significant use in animal and human treatment regimens, but also has many other practical uses, including many in vitro uses. Certain of these uses relate to the specific binding properties of the antibody or immune complex. All those compounds of the invention include at least one antibody component, which may be used in virtually all binding embodiments in which the first antibody may be used.

The presence of an attachment agent, if relevant, provides advantageous properties, but does not rule out the use of the first antibody domain in any binding assay. Thus, suitably useful binding assays include those commonly used in the art, such as immunoblots, western blots, dot blots, RIA, ELISA, immunohistochemistry, as further described herein. , Fluorescent labeling cell sorting (FACS
), Immunoprecipitation, affinity chromatography, etc.).

Certain standard binding assays involve immobilizing the antigen on a solid support matrix (eg, nitrocellulose, nylon or a combination thereof) (eg, as in immunoblots, Western blots and related assays). A) Assay. Another important assay is an ELISA. All such assays are VEGF-like, as can be applied in the diagnosis of angiogenic diseases.
Can be easily adapted for use in the detection of The agents of the present invention may also be used in immunohistochemistry; in fluorescence-activated cell sorting, flow cytometry or flow microfluorimetry; in immunoprecipitation; in antigen purification embodiments (eg, affinity chromatography (for bispecific antibodies). Fresh frozen paraffin embedding in many other binding assays known to those of skill in the art given the information presented herein); It can be used with both tissue blocks and formalin fixed paraffin embedded tissue blocks.

A further practical use of the present antibodies is as a control in a functional assay. These include many in vitro and ex vivo assays and systems and animal model studies. If the binding and functional properties of the antibodies of the invention are particularly specific (ie they are VEGF (but not VEGFR1) VEG)
Inhibiting binding to FR2 and signaling through VEGFR2), such "control" applications are indeed very useful. Assays that would benefit from such a practical application of the invention include, for example, assays related to VEGF-mediated endothelial cell proliferation, VEGF-induced phosphorylation and VEGF-induced vascular permeability, and corneal micropocket assays of neovascularization and Chicken chorionic allantois (
CAM) assay. These assay systems have also evolved into in vitro or ex vivo drug screening assays, where the present provision of biological materials with well-defined properties is of particular importance.

C. Immunoconjugates The present invention provides highly effective naked or unconjugated antibodies for use in anti-angiogenic methods, but with VEGFR2 blocking antibodies (anti-VEGF antibodies) or 2
C3-based immunoconjugates, immunotoxins and coaguland (coagul)
igand) are also provided herein. VEGFR2 blocking antibody (anti-V
Currently preferred agents for use in therapeutic conjugates based on (EGF antibodies) or 2C3 are radiotherapeutic agents (as exemplified by the radiodiagnosis disclosed herein), anti-angiogenic agents, apoptosis induction Agents, anti-tubulin agents, anti-cellular or anti-cytotoxic agents and clotting agents (coagulation factors).

To produce immunoconjugates, immunotoxins and coag ligands, recombinant expression can be used to make fusion proteins as is known to those of skill in the art and as further disclosed herein. . Similarly, immunoconjugates, immunotoxins and coaguligands can be produced using avidin: biotin cross-linking or any chemical conjugation and cross-linking techniques developed for antibody conjugates.

C1. Toxins and Anticellular Agents For certain applications, therapeutic agents are cytotoxic or pharmacological agents that have the ability to eliminate or inhibit endothelial cell proliferation or cell division. Especially cytotoxic drugs, cytostatic drugs or else anticellular drugs. In general, these aspects of the invention contemplate the use of any pharmacological agent that blocks VEGFR2, is conjugated to an anti-VEGF antibody or 2C3-like antibody, and can be delivered to the targeted endothelium in an active form. I do.

[0435] Exemplary anticellular agents include chemotherapeutic agents and cytotoxins. Chemotherapeutic agents that may be used include: hormones (eg, steroids); antimetabolites (eg, cytosine arabinoside, fluorouracil, methotrexate or aminopterin); anthracyclines; mitomycin C; vinca alkaloids; demecortin Etoposide: mitramycin: an antitumor alkylating agent (eg, chlorambucil or melphalan). Other embodiments may include agents such as cytokines. Basically, it can be successfully bound to or associated with an antibody in a manner that allows targeting, internalization, release and / or presentation to blood components at the site of the targeted endothelial cell Any anticellular agent can be used as long as it can be done.

For example, if the target antigen does not internalize in a manner consistent with efficient intoxication by toxic compounds, chemotherapeutic agents (eg, anti-tumor drugs, cytokines, antimetabolites, alkylating agents, hormones, etc.) may be used. There may be situations where it is desired to target. Various chemotherapeutic and other pharmacological agents, including doxorubicin, daunomycin, methotrexate, vinblastine, neocarzinostatin, macromycin, trenimon and α-amanitin, are now successfully bound to antibodies. And has been shown to function pharmacologically.

In other situations, any potential side effects from cytotoxin-based therapy may be
A synthesis inhibitors (eg, daunorubicin, doxorubicin, adriamycin, etc.) can be removed. Thus, these agents are preferred examples of anti-cellular agents for use in the present invention.

With respect to cytostatic agents, such compounds generally interfere with the natural cell cycle of the target cell, preferably so that the cell exits the cell cycle. Exemplary cytostatic agents include.

[0439] Can be conjugated to a VEGFR2 blocking anti-VEGF antibody or a 2C3-based antibody,
A wide variety of negatives are known. Examples include many useful plant, fungal or bacterial toxins, to name a few: various A-chain toxins, especially ricin A-chain; ribosome inactivation, to name a few. Α-sarcin; aspergillin; restrictocin; ribonuclease (eg, placental ribonuclease); diphtheria toxin; and pseudomonas exotoxin.

Of the toxins, ricin A chain is preferred. Most preferred toxin moieties for use in connection with the present invention are toxin A chains that have been treated to modify or remove carbohydrate residues.
It is the so-called deglycosylated A chain (dgA). The deglycosylated ricin A chain is
It is preferred because of its extreme effectiveness, longer lifespan, and because it is economically feasible to manufacture at clinical grade and scale.

It may be desirable from a pharmacological point of view to use the smallest molecule but nonetheless capable of providing an appropriate biological response. For example,
It may be desirable to use smaller A-chain peptides that provide a sufficient anti-cell response. To this end, it has been discovered that the ricin A chain can be "truncated" by removal of the 30 N-terminal amino acids by Nagarase (Sigma) and still retain sufficient toxin activity. It is proposed that, if desired, this truncated A chain can be used in a conjugate according to the invention.

Alternatively, it can be appreciated that the application of recombinant DNA technology to the toxin A chain portion provides additional benefits according to the present invention. A modification that is now a smaller or other mode of variant peptide in that the cloning and expression of a biologically active ricin A chain has been achieved, but nevertheless exhibits adequate toxin activity. It is possible to identify and prepare body peptides. Furthermore, the fact that the ricin A chain has now been cloned allows the application of site-directed mutagenesis, through which A chain derived peptides can be easily prepared and screened, and combined with the present invention. Further useful parts are obtained for all uses.

C2. Coagulation Factors VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies of the invention can be linked to components that can directly or indirectly stimulate coagulation to form coag ligands. Here, the antibody can be linked directly to the coagulant or coagulation factor, or can be linked to a second binding region that binds the coagulant or coagulation factor and then releases them. As used herein, the terms “coagulant” and “coagulation factor” are each used to coagulate when provided under appropriate conditions, preferably in a particular in vivo environment (eg, tumor vasculature). Refers to a component that can directly or indirectly stimulate

[0444] Preferred coagulation factors are tissue factor compositions (eg, truncated TF (tTF
), Dimeric TF molecules, multimeric TF molecules and mutant TF molecules). "
“Truncated TF” (tTF) refers to a TF structure that has been made by removing sufficient amino acid sequence to cause a change in this property in a membrane-associated defect. In this situation, "sufficient amount" means TF
The amount of transmembrane amino acid sequence originally sufficient to allow the molecule to enter the membrane or otherwise mediate functional membrane binding of the TF protein. Thus, removal of such a "sufficient amount of transmembrane sequence" results in a truncated tissue factor protein or polypeptide lacking phospholipid membrane binding capacity, so that the protein is substantially free of phospholipids. A soluble protein that does not bind significantly to lipid membranes. Therefore,
Truncated TF is substantially incapable of converting factor VII to factor VIIa in a standard TF assay, and still involves activating factor X in the presence of factor VIIa, the so-called Retains catalytic activity.

US Pat. No. 5,504,067 is specifically incorporated herein by reference for the purpose of further describing such truncated tissue factor proteins. Preferably, the tissue factor for use in these aspects of the invention comprises:
Generally, the transmembrane and cytoplasmic regions of the protein (amino acids 220-263)
Lacks. However, the truncated TF molecule need not be limited to molecules of the correct length of 219 amino acids.

A tissue factor composition may also be useful as a dimer. Any truncated tissue factor construct, mutated tissue factor construct, or other tissue factor construct is
It can be prepared in dimeric form for use in the present invention. As known to those skilled in the art, such TF dimers can be prepared by using standard techniques of molecular biology and recombinant expression. Here, the two coding regions are prepared in frame and expressed from an expression vector. Similarly, various chemical conjugation techniques can be used in combination with the preparation of the TF dimer. Each TF monomer is
It can be derivatized before conjugation. All such techniques are readily known to those skilled in the art.

If desired, the tissue factor dimer or multimer can be attached via a biologically-releasable linkage (eg, a selectively cleavable linker or amino acid sequence). For example, peptide linkers that include cleavage sites for enzymes that are preferentially located or active in the tumor environment are contemplated. Exemplary forms of such a peptide linker are peptide linkers that are cleaved by urokinase, plasmin, thrombin, factor IXa, factor Xa or a metalloproteinase (eg, collagenase, gelatinase or stromelysin).

In certain embodiments, the tissue factor dimer further comprises a hydrophobic membrane that is interrupted to enhance the functional association of the tissue factor with the phospholipid membrane (under certain predetermined conditions only). It may include an insert. When described in the context of truncated tissue factor, a hydrophobic membrane-associated sequence is generally a stretch of amino acids that, due to their hydrophobic nature, promotes association with the phospholipid environment. Similarly, fatty acids can be used to provide a potential membrane insert.

[0449] Such membrane inserts can be located at either the N-terminus or C-terminus of the TF molecule, as long as their attachment does not interfere with the functional properties of the TF construct, or in general, any other Can be added. The significance of the hindered insert is that it remains non-functional until the TF construct is located in the tumor milieu, and the hydrophobic addition
To be accessible and still further facilitate physical association with the membrane. Again, biologically releasable bonds and selectively cleavable sequences are particularly useful in this regard, where only the bonds or sequences are localized in the tumor environment and certain enzymes or other biological activities It is intended to be cut or otherwise altered upon exposure to the molecule.

In other embodiments, the tTF construct can be a multimer or a polymer. In this context, a "polymer construct" includes three or more tissue factor constructs. A "multimeric or polymeric TF construct" is a construct comprising at least a first TF molecule or derivative operably linked to a second and third TF molecule or derivative. Multimers may contain from about 3 to about 20 such TF molecules. Individual TF units within a multimer or polymer can also be linked by a selectively cleavable peptide linker or other biologically releasable linkage, if desired. Again, for the TF dimers discussed above, the constructs can be easily made using either recombinant manipulation and expression, or using standard synthetic chemistry.

Still further TF constructs useful in the context of the present invention are mutants that lack the ability to activate factor VII. Such "Factor VII activating variants" generally bind a functional Factor VII / VIIa and proteolytically activate Factor X, but activate Factor VII proteolytically. TF variants are defined herein as having substantially no ability to transform. Thus, such a construct is a TF variant that lacks factor VII activating activity.

The ability of such Factor VII activating mutants to function in promoting tumor-specific coagulation is their specific delivery to tumor vasculature and low levels of Factor VIIa in plasma. Based on the presence of factors. Upon administration of such a Factor VII-activating mutant targeting agent conjugate, the mutant is localized within the vasculature of the angiogenic tumor. Prior to localization, the TF variant generally cannot promote coagulation in any other body part based on its inability to convert factor VII to factor VIIa. However, upon localization and accumulation within the tumor area, the mutant then encounters sufficient Factor VIIa from the plasma to initiate the extrinsic coagulation pathway, leading to tumor-specific thrombosis. Exogenous Factor Vila may also be administered to the patient.

Any one or more of the various Factor VII activating variants can be prepared and used in combination with the present invention. There is a significant amount of scientific knowledge regarding the recognition site on the TF molecule for Factor VII / VIIa. Thus, it is understood that the VII activation region is generally located between about amino acid 157 and about amino acid 167 of the TF molecule. However, residues outside this region may also be found to be involved in VII activating activity, and thus may include any one or more of the residues commonly located from about amino acid 106 to about amino acid 209 of the TF sequence. It may be contemplated to introduce mutations (WO94 / 07515; WO94 / 28017; each of which is incorporated herein by reference).

A variety of other clotting factors can be used in combination with the present invention, as exemplified by the agents set forth below. Thrombin, factor V / Va and derivatives, factor VIII / VIIIa and derivatives, factor IX / IXa and derivatives, factor X / Xa and derivatives, factor XI / XIa and derivatives, factor XII / XI
Factor Ia and derivatives, factor XIII / XIIIa and derivatives, factor X activator and factor V activator can be used in the present invention.

The Russell's viper snake venom Factor X activator is contemplated for use in the present invention. Monoclonal antibodies specific for the factor X activator present in Russell's viper venom have also been produced and can be used to specifically deliver the factor as part of a bispecific binding ligand.

Thromboxane A ₂ is formed from endoperoxide by the continuous action of the enzymes cyclooxygenase and thromboxane synthetase in platelet microsomes. Thromboxane A ₂ is readily produced by platelets and is a potent vasoconstrictor because of its ability to produce platelet aggregation. Thromboxane A ₂ and active analogues are contemplated for use in the present invention.

Thromboxane synthase, and other enzymes that synthesize platelet-activating prostaglandins, can also be used as “coagulants” in the context of the present invention. Monoclonal antibodies to thromboxane synthase, and immunoaffinity purification of thromboxane synthase are known; so is the cDNA for human thromboxane synthase.

Α2-antiplasmin, an α2-plasmin inhibitor, is a naturally occurring proteinase inhibitor in human plasma that functions to efficiently inhibit lysis of fibrin clots induced by plasminogen activator It is. α2-antiplasmin is a particularly potent inhibitor and is contemplated for use in the present invention.

Where cDNA sequences for α2-antiplasmin are available, recombinantly expressed and / or fusion proteins are preferred. Monoclonal antibodies to α2-antiplasmin are also available, which can be used in the bispecific binding ligand embodiments of the invention. These antibodies can be used to deliver exogenous α2-antiplasmin to the target site, or to collect endogenous α2-antiplasmin and concentrate it within the target area.

C3. Anti-Tubulin Drugs A series of drugs exert their effects through interference with tubulin activity. Certain "anti-tubulin drugs" are potent chemotherapeutic agents, as tubulin function is essential for mitosis and cell viability. Some well-known and currently preferred anti-tubulin agents for use with the present invention are: colchicine; taxanes (eg, taxol); vinca alkaloids (eg, vinblastine, vincristine and vindestine); and combretastatin ( c
ombrestatatin). Other suitable anti-tubulin agents include cytochalasins (including B, J, E), dolastatin, auristatin PE, paclitaxel, ustiloxin D, rhizoxin, 1069C85.
, Colcemid, albendazole, azatoxin (aza)
toxin) and nocodazole.

US Pat. Nos. 5,892,069, 5,504,074 and 5
Combretastatins are generally estradiol derivatives that inhibit cell mitosis, as described in U.S. Pat. No. 6,661,143, each of which is hereby incorporated by reference in detail. Exemplary combretastatins that may be used in combination with the present invention include combretastatins based on combretastatins A, B and / or D and U.S. Patent Nos. 5,892,069 and 5,504. , 07
No. 4 and No. 5,661,143. Combretastatin A-1, A-2, A-3, A-4, A-5, A-6
, B-1, B-2, B-3 and B-4 are examples of the above type.

US Pat. Nos. 5,569,786 and 5,409,953 disclose combretastatins A-1, A2, A-3, B-1, B-2, B-3 and B-. For the purpose of describing the isolation, structural characterization and synthesis of each of the No. 4 and formulations and methods for treating neoplastic growth using such combretastatins, Incorporated as a reference. Any one or more such combretastatins can be used in combination with the present invention.

US Pat. Nos. 5,892,069, 5,504,074 and 5,66
Combretastatin A-4 may also be used with the present invention, as described in 1,143 and 4,996,237, each of which is incorporated herein by reference in detail. . U.S. Patent No. 5,561,112 discloses herein a series of suitable combretastatin A-4 prodrugs, which are intended to be used in combination with the present invention. Incorporated as a reference.

US Pat. No. 4,940,726, which is hereby incorporated by reference in its entirety, describes macrocyclic lactones designated combretastatin D-1 and “combretastatin D-2”. Which may be used in combination with the compositions and methods of the present invention. U.S. Patent No. 5,430,062, which is hereby incorporated by reference in its entirety, relates to stilbene derivatives and combretastatin analogs having anticancer activity, which may be used in combination with the present invention.

C4. Anti-Angiogenesis Agents The invention specifically provides for combined anti-angiogenesis. Angiopoietin is VEG
Like members of the F family, it is a growth factor specific for vascular endothelium (Dav
is and Yancopoulos, 1999: Holash et al., 1999; incorporated herein by reference). The first described angiopoietin is a naturally occurring receptor activator or agonist, angiopoietin-1 (
Ang-1; SEQ ID NO: 1 and SEQ ID NO: 2), and the naturally occurring receptor antagonist angiopoietin-2 (Ang-2; SEQ ID NO: 3 and SEQ ID NO: 4). Both are endothelial cell tyrosine kinase receptors Tie
Acts by 2.

Two new angiopoietins, angiopoietin-3 (mouse) and angiopoietin-4 (human), have also been identified (Valenzuela et al.
1999). Angiopoietin-3 appears to act as an antagonist (such as Ang-2), while angiopoietin-4 appears to function as an agonist (such as Ang-1) (Valenzuela et al., 1999)
. A protein called angiopoietin-3 has also been cloned from human heart and reported to have no mitogenic effect on endothelial cells (K
im et al., 1999).

[0467] VEGF is required for early stages of vascular development, while angiopoietin-1 is generally required for later stages of angiogenesis. Therefore, VEGF
Acts to promote endothelial cell differentiation, proliferation and early angiogenesis. Angiopoietin-1 acts via the Tie2 receptor to promote maintenance and stabilization of the mature duct. Thus, angiopoietin-1 is a maturation or stabilizing factor, which converts immature blood vessels into mature blood vessels by promoting the interaction between endothelial cells and surrounding supporting cells. (Holash et al., 19
99).

Angiopoietin-1 enhances revascularization in ischemic tissue (Sh
yu et al., 1998), and increasing the survival of vascular networks exposed to either VEGF or aFGF forms (Papapetropoulos et al., 199).
9) is shown. These authors have also shown that angiopoietin-1 prevents apoptotic death in HUVECs induced by withdrawal of the same form of aFGF (Papapetropoulos et al., 1999). Such data is consistent with the direct role of angiopoietin-1 on human endothelial cells and its interaction with other angiogenic molecules that stabilize vasculature by promoting the survival of differentiated endothelial cells. I do.

Since angiopoietin-1 transmits maturation and stability signals, we have carefully considered aspects of the invention relating to delivery of target angiopoietin-1. The present inventors inferred that angiopoietin-1 is a maturation factor and thus renders tumor vessels growth factor independent. Accordingly, one aspect of the invention is a non-targeted angiopoietin for consolidating the VEGF non-responsive properties of a target tube.
1 regarding the use.

It is inferred that the use of tumor binding ligands to deliver angiopoietin-1 molecules to tumor blood vessels will readily deliver on the order of 500,000 angiopoietin molecules to the lumen. This overwhelms the Tie2 receptor system and as a whole Ti
Saturate the e2 receptor with angiopoietin-1 ligand. Thus, the combined effect of angiopoietin-2 and VEGF (see discussion below) is inhibited because angiopoietin-2 cannot bind.

Delivery of angiopoietin-1 to tumors, preferably to the tumor vasculature, can also be used with various other anti-cancer strategies, as described in detail herein. Achieve a combined therapeutic effect. A typical response of a tumor to a treatment that induces at least some necrosis is to initiate vascular regeneration. The signals of angiopoietin-1 cannot remove them because they direct blood vessels to maturation and cannot compensate for the loss of tumor mass induced by early therapeutic agents. Thus, these observations make another preferred embodiment of the invention of angiopoietin delivery (ie, the combined use of angiopoietin-1 targeting in combination with any one or more anti-cancer agents, including conventional chemotherapeutic agents). Provide aspects.

The effect of angiopoietin-1 on delivery is essentially due to the vascular remodeling function (re
modelling). Whether used alone or in combination with therapeutic agents, the value of angiopoietin-1 targeting is particularly enhanced by the inherent safety of this therapeutic approach. There are no significant disadvantages for angiopoietin-1 treatment. Even in the unlikely event that a certain amount of targeted Ang-1 is incorrectly given to non-tumor tissue, all that follows is that the vasculature in the target area becomes more stable and / or Or go into hibernation. In this regard, Ang-1 may also be used as an anti-inflammatory.

Angiopoietin-2 is useful for use in tumor-targeted therapy, particularly for VEGF of the invention.
It is currently a preferred agent for use in combination with inhibition. However, due to the unique effects of angiopoietin 2 under different conditions, especially at varying levels of VEGF, we have carefully revisited aspects of the invention relating to targeted angiopoietin-2 delivery.

Angiopoietin-2 is also a ligand for the Tie2 receptor, but generally neutralizes vascular maturation / stabilization mediated by angiopoietin-1. Thus, it is an angiopoietin-1 antagonist and acts to disrupt the capillary structure. Under appropriate conditions, angiopoietin-2 transmits a negative signal to target cells, and the destabilization induced by angiopoietin-2 results in vascular regression. This is the first of angiopoietin-2 to be exploited in the targeted delivery of angiopoietin-2 to tumors, preferably tumor vasculature.
It is a feature of. Sufficient angiopoietin-2 to simply deliver, which is easily achievable using a VEGFR2-blocking anti-VEGF antibody such as 2C3,
Overwhelms any other signals that may be present in the tumor environment and promotes vascular regression. Since there is a carefully controlled interaction between angiopoietin in the natural environment, the abnormal bias of the system in favor of regression is due to the constant angiopoietin-2 signaling resulting in angiopoietin-1 and VEGF Both effects can be eliminated.

Thus, the use of tumor targeting angiopoietin-2 alone may be used to advantage to induce tumor vascular regression, especially as an early mechanism of therapeutic intervention. However, in general, destabilization induced by angiopoietin-2 can result in either vascular regression or regeneration. In the absence of other angiogenic stimuli, particularly in the absence of VEGF, destabilization leads to vascular regression; whereas, in the presence of high levels of VEGF, destabilization occurs. Promotes the angiogenic response (Holas
h et al., 1999).

Vessels that undergo destabilization in response to angiopoietin-2 can be "rescued" from regression by exposure to other stimuli. Thus, angiopoietin-2 renders endothelial cells responsive to other angiogenic stimuli and promotes angiogenic responses under defined conditions. In particular, VEGF can proliferate ang-2 destabilized cells and form early neovasculature (Asahara et al., 1998; Holash et al., 1999). Indeed, angiopoietin-2 expression in tumor tissue has been reported (Tanaka et al., 1999), where it was presumed to act in combination with VEGF to promote angiogenesis (Stratmann et al.). , 19
98). Angiogenesis initiated by angiopoietin-2 and VEGF
These molecules are made "co-angiogenic".

An integrated model to explain the positive and negative effects of angiopoietin-2 on blood vessels in certain tumor types has been previously reported (Holash et al., 1999). In this model, angiopoietin-
2-induced destabilization selects vessels from the surrounding host vasculature (cooling)
) First causes significant vascular regression in the tumor. The high levels of angiopoietin-2 produced by endothelial cells associated with tumors negate the survival signal clearly produced by the low level, constitutive expression of angiopoietin-1. Thus, angiopoietin-2 marks vessels selected for regression of apoptosis (Holash et al., 1999). However, in spite of the resulting tumor necrosis, surviving tumor cells are not treated with VEG to ensure their survival.
Up-regulate F. VEGF abolishes the regression signal from angiopoietin-2 and indeed promotes angiogenesis (Holash et al., 19
99), then co-expression of VEGF and angiopoietin-2 results in robust angiogenesis in the tumor periphery.

Although seemingly inconsistent, the effects of angiopoietin-2 can be explained based on other existing signals, particularly VEGF, and many can be expected. Angiopoietin-2 in the absence of another angiogenic signal
Destabilizes the vessels and makes them immature, leading to regression. Stimulation, especially VEGF
In the presence of an angiopoietin-2, the instability caused by angiopoietin-2 actually results in angiogenesis when the vessel is "prepared" to receive a secondary angiogenic stimulus. Thus, it is believed that the angiogenic effects of a number of modulators are achieved, at least in part, by modulation of the autocrine loop of angiopoietin-2 activity in microvascular endothelial cells (Mandoriota and Pepper,
1998).

The dual biological role of angiopoietin-2 has led us to develop additional therapeutic strategies that account for other signals in the tumor environment, particularly VEGF. Since angiopoietin-2 and VEGF cooperatively stimulate angiogenesis, a preferred aspect of the invention uses tumor-targeted angiopoietin-2 delivery in combination with the VEGF-inhibiting antibodies of the invention. Thus, angiopoietin-
2 works in retraction and does not work in angiogenesis.

In view of the above description, the present invention provides a VEGFR2-blocking anti-VEGF antibody (eg, 2C3), which comprises angiopoietin-1, angiopoietin-
2. It is understood that it is operably linked to, or otherwise functionally related to, any one or more of angiopoietin-3 and / or angiopoietin-4. An exemplary angiopoietin-1 composition is SEQ ID NO: 1 (DNA) and SEQ ID NO: 2 (protein), while an angiopoietin-2 composition is SEQ ID NO: 3 (DNA) and SEQ ID NO: 4 (protein ). Angiopoietin-3 is an antagonist and is commonly used like angiopoietin-2; the agonist angiopoietin-4 can be used in the manner of angiopoietin-1. Valenzuela et al. (1999) is specifically incorporated by reference for the purpose of further supplementing the teachings herein of angiopoietin-3 and angiopoietin-4.

Further, fusion proteins of angiopoietin are also envisioned for use in the present invention. One example is the stable Ang-l- included herein as SEQ ID NO: 5.
Ang-2 fusion protein. This protein contains the first 73 residues of angiopoietin-2, up to the DAPLEY sequence, fused to the angiopoietin-1 sequence beginning at amino acid 77. It also has a mutation at position 265 in the angiopoietin-1 sequence, where Cys is replaced with Ser.

Other anti-angiogenic agents for use with the present invention include angiostatin and endostatin. Angiostatin is disclosed in US Pat. No. 5,776.
No. 5,704,725 and 5,733,876. Each of these is incorporated herein by reference. Angiostatin is a protein having a molecular weight of about 38 kDa to about 45 kDa, as determined by reducing polyacrylamide gel electrophoresis,
It contains approximately the kringle regions 1-4 of the plasminogen molecule. Angiostatin generally has an amino acid sequence substantially similar to a fragment of mouse plasminogen starting at amino acid number 98 of the intact mouse plasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species. For example, in human angiostatin, this amino acid sequence is substantially similar to the sequence of the mouse plasminogen fragment described above, but the active human angiostatin sequence is identical to amino acid number 97 of the intact human plasminogen amino acid sequence. Or start at either 99. In addition, human plasminogen has similar anti-angiogenic activity and can be used as shown in mouse tumor models.

Since angiostatin and endostatin are the first angiogenesis inhibitors in mice to demonstrate the ability to not only inhibit tumor growth but also effect tumor regression, they are the focus of intense research. Become. Elastase, macrophage metalloelastase (MME), matrilysin (matrilysin)
) (MMP-7), and 92 kDa gelatinase B / type IV collagenase (M
There are multiple proteases that have been shown to produce angiostatin from plasminogen, including MP-9).

MME can produce angiostatin from plasminogen in tumors,
Granulocyte macrophage colony stimulating factor (GMCSF) then upregulates MME expression by macrophages that induce angiostatin production. The role of MME in angiostatin production is supported by the finding that MME is indeed expressed in clinical samples of hepatocellular carcinoma from patients. Another protease that could produce angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown to generate an angiostatin-like fragment from plasminogen in vitro.
The mechanism of action for angiostatin is currently unclear,
It is presumed to bind to an unidentified cell surface receptor on endothelial cells that induces them to undergo programmed cell death or mitosis.

Endostatin appears to be an even more potent anti-angiogenic and anti-tumor agent, and is particularly preferred for linking to VEGFR2-blocking anti-VEGF antibodies (eg, 2C3). Endostatin is effective in causing regression in many tumor models in mice. The tumors do not develop resistance to endostatin, and after multiple cycles of treatment, the tumors enter a dormant state, during which they do not increase in volume. In this dormant state, the proportion of tumor cells undergoing apoptosis increased and resulted in a population that remained essentially the same size.

US Pat. No. 5,854,205 to Folkman and O'Reiley et al.
No. (incorporated herein by reference in detail) relates to endostatin and its use as inhibitors of endothelial cell proliferation and angiogenesis. Endostatin protein corresponds to the C-terminal fragment of collagen type XVIII, and this protein can be isolated from a variety of sources. US Patent 5,854
No. 205 also states that endostatin is a collagen XVIII, collagen X
It teaches that it may have the amino acid sequence of type V, or a fragment of BOVMPE1 progastrin esterase. Combinations of endostatin with other anti-angiogenic proteins, particularly angiostatin, are also described in US Pat. No. 5,854,205, so that the combined composition provides Can be effectively retracted.

[0488] Endostatin and angiostatin are preferred agents for tumor delivery according to the present invention. Vasculostatin, canstatin and maspin are also preferred agents. Endostatin is one of the most preferred drugs. Endostatin 2C3 fusion proteins can be prepared as described herein.
Various forms of the chemically linked Endostatin-2C3 construct are also described in the present application.

(C5. Apoptosis Inducing Agent) The present invention also induces apoptosis in any cells in a tumor, including tumor cells and tumor vascular endothelial cells. Although many anti-cancer agents may have apoptosis-inducing effects as part of their mechanism of action, certain agents may be discovered using this as the primary mechanism, as described below, Designed or selected.

[0490] Mutations in tumor suppressor genes (eg, p53) have been reported in many forms of cancer. Inactivation of p53 results in failure to promote apoptosis. This deficiency causes cancer cells to progress to tumor formation rather than being fated to cell death. Thus, delivery of tumor suppressors is also contemplated for use in the present invention to stimulate cell death. Exemplary tumor suppressors include p53, retinoblastoma gene (Rb), Wilms tumor (WT1), baxα, interleukin-
1b-converting enzyme and family, MEN-1 gene, neuroblastoma type I (NF
1), cdk inhibitor p16, colorectal oncogene (DCC), familial adenomatous polyposis gene (FAP), multiple tumor suppressor gene (MTS-1), BRC
A1 and BRCA2 include, but are not limited to.

Preferred for use is p53 (US Pat. Nos. 5,747,469; 5,677,178; and 5,756,455; each of which is incorporated herein by reference). Retinoblastoma, BRCA1 (US Pat. No. 5,750).
No. 5,654,155; No. 5,710,001; No. 5,756,294; No. 5,709,999; No. 5,693,473.
No. 5,753,441; No. 5,622,829; and No. 5,7
No. 47,282; each of which is incorporated herein by reference), MEN-1 (
GenBank Accession No. U93236) and the gene for adenovirus E1A (US Pat. No. 5,776,743; incorporated herein by reference).

Other compositions that can be delivered by a VEGFR2 blocking anti-VEGF antibody (eg, 2C3) include genes encoding tumor necrosis factor-related apoptosis-inducing ligand (referred to as TRAIL), and TRAIL polypeptides (US Pat. Fifth
No. 763,223; incorporated herein by reference); US Pat. No. 5,60.
5,826 (incorporated herein by reference) 24 kD apoptosis-associated protease; Fas-associated factor 1, FAF1 (US Pat. No. 5,750,653; incorporated herein by reference). No. Interleukin-1β
The provision of converting enzymes and family members, which are also reported to stimulate apoptosis, are also contemplated for use in these aspects of the invention.

The following compounds may also be used; carbostyril
ril) derivatives (U.S. Pat. Nos. 5,672,603; and 5,464,8).
33; each of which is incorporated herein by reference); Branched apopogenic peptides (US Pat. No. 5,591,717; incorporated herein by reference). Phosphotyrosine inhibitors and non-hydrolyzable phosphotyrosine analogs (US Pat. Nos. 5,565,491;
93,627; each of which is incorporated herein by reference); RXR retinoid receptor agonists (US Pat. No. 5,399,586; incorporated herein by reference); , Antioxidants (US Pat. No. 5,571,523)
No .; incorporated herein by reference). Tyrosine kinase inhibitors (eg, genistein) can also be linked to agents of the invention that target cell surface receptors (VEGFR1) (see US Pat. No. 5,587,459, incorporated herein by reference). As supported).

C6. Biologically Functional Equivalents Equivalents or further modifications of 2C3-based antibodies or any other VEGFR2-blocking anti-VEGF antibodies are now generally starting from the materials provided above. And can be made as Modifications and changes can be made in the structure of such antibodies, and still obtain molecules with similar or other modes of desired characteristics.
For example, certain amino acids may have interactive binding capabilities (eg, aminophospholipids, PS
And binding to PE) can be substituted for other amino acids in the protein structure without significant loss. These considerations also include toxins, anti-angiogenics,
This applies to apoptosis-inducing agents and coagulants.

Because it is the ability and nature of the protein to define the biological functional activity of the protein, certain amino acid sequence substitutions are made in the protein sequence (or, of course, the underlying DNA sequence). It is possible to obtain proteins which have similar (agonist) properties. Accordingly, various changes may occur in the antibody or therapeutic agent sequence (without appreciable loss of their biological utility or activity).
Or the underlying DNA sequence). Biological functional equivalents made from mutating the underlying DNA sequence can be obtained using the codon information provided in Table A herein and supporting technical details for site-directed mutagenesis. Can be done.

The notion that there is a limit to the number of changes that can be made within a given portion of a molecule and still result in molecules having acceptable levels of equivalent biological activity is described in “Biological Function Also being specific to the definition of a "sex equivalent" protein or peptide,
It is well understood by those skilled in the art. Accordingly, biologically functional equivalent proteins and peptides are defined herein as proteins and peptides in which little or all, but not all, of the particular amino acids can be substituted. It will be appreciated that multiple different proteins / peptides having different substitutions can be readily made and used in accordance with the present invention.

Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substitutions (eg, their hydrophobicity, hydrophilicity, charge, size, etc.). Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; alanine, glycine and serine are all of similar size; and phenylalanine, tryptophan And tyrosine are all generally shown to be of similar shape. Thus, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are defined herein as biologically functional equivalents.

In making more quantitative changes, the hydropathic index of amino acids can be considered. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics, which are: isoleucine (+4.5); valine (+4.
2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); -0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (
Glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-3.2); -4.5).

The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Dooitt).
le, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still retain similar biological activity. In making a change based on the hydropathic index, substitution of amino acids whose hydropathic index is within ± 2 is preferred, substitutions within ± 1 are particularly preferred, and substitutions within ± 0.5 are even more preferred. Particularly preferred.

Thus, it is understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent protein. As detailed in U.S. Pat. No. 4,554,101, which is incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0 ); Lysine (+3.0); aspartic acid (+ 3.0 ± 1); glutamic acid (+ 3.0 ± 1); serine (+0.3); asparagine (+0.2)
Glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); 1.0); methionine (-1.3); valine (-0.5); leucine (
-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

In making a change based on a hydrophilicity value, substitutions of amino acids whose hydrophilicity values are within ± 2 are preferred, those that are within ± 1 are particularly preferred, and those that are within ± 0.5. Is even more particularly preferred.

C7. Fusion Proteins and Recombinant Expression VEGFR2-blocking anti-VEGF antibodies or 2C3-based immunoconjugates of the invention can be readily prepared as fusion proteins using molecular biology techniques. Any fusion protein can be designed and made using any of the therapeutic agents described herein and known in the art. Fusion protein technology is readily adapted to prepare fusion proteins in which the two portions are joined by a selectively cleavable peptide sequence. Currently preferred fusion proteins are fusion proteins that include endostatin. Endostatin, when accompanied by any other therapeutic agent, can be linked to the terminal end of the antibody or to any other point apart from the CDRs. Therapeutic agents such as endostatin can also be prepared "integral", where they are preferably associated with a selectively cleavable peptide to allow release of the drug after targeting.

The use of recombinant DNA technology to achieve such goals is now a standard procedure for those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo / genetic recombination. Furthermore, D
NA and RNA synthesis can be performed using an automated synthesizer (eg,
See the technique described in Sambrook et al., 1989, incorporated herein by reference).

The preparation of such fusion proteins generally involves the preparation of first and second DNA coding regions, and the preparation of a single coding region encoding the desired fusion protein. It involves operatively linking or joining regions in frame. In the context of the present invention, VEGFR2 blocking anti-VEGF antibodies or 2C
The DNA sequence of the 3-like antibody is ligated in frame with the DNA sequence encoding the therapeutic agent. Which part of the construct is prepared as an N-terminal region or a C-terminal region is generally not considered particularly relevant.

[0502] Once the desired coding region has been generated, an expression vector is created. The expression vector contains one or more promoters, upstream of the inserted DNA region, that act to promote transcription of the DNA, and thus act to promote expression of the encoded recombinant protein. This is the meaning of “recombinant expression”.

To obtain a so-called “recombinant” version of a VEGFR2-blocking anti-VEGF antibody or 2C3-based immunoconjugate, it is expressed in recombinant cells. Manipulation of DNA segments for expression in prokaryotic or eukaryotic systems can be performed by techniques generally known to those of skill in recombinant expression. It is contemplated that virtually any expression system can be used in expressing the VEGFR2-blocking anti-VEGF antibody or 2C3-based immunoconjugate construct.

[0507] Such proteins can be successfully expressed in eukaryotic expression systems, such as CHO cells. However, E. Bacterial expression systems such as E. coli pQE-60 are believed to be particularly useful for large-scale preparation and subsequent purification of VEGFR2-blocking anti-VEGF antibodies or 2C3-based immunoconjugates. The cDNA is also expressed in bacterial systems, and the encoded protein expressed can be expressed as a fusion with β-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase, and the like. Bacterial expression is considered to have advantages over eukaryotic expression in terms of ease of use and the quality of the material obtained therefrom.

For microbial expression, US Pat. No. 5,583,013, for the purpose of still further supplementing the present disclosure in connection with expression of the gene in recombinant host cells;
Nos. 5,221,619; 4,785,420; 4,704,36
No. 2; and 4,366,246 are incorporated herein by reference.

Recombinantly produced VEGFR2-blocking anti-VEGF antibodies or 2C3-based immunoconjugates can be purified and formulated for human administration. Alternatively, the nucleic acid encoding the immunoconjugate can be delivered through gene therapy. Although naked recombinant DNA or plasmids can be used, the use of liposomes or vectors is preferred. The ability of certain viruses to enter cells via receptor-mediated endocytosis, and to integrate into the host cell genome and stably and efficiently express viral genes, makes them compatible with foreign genes in mammalian cells. Makes it an attractive candidate to populate. Preferred gene therapy vectors for use in the present invention are generally viral vectors.

[0510] Retroviruses have their ability to integrate their genes into the host genome, their ability to transfer large amounts of foreign genetic material, their ability to infect a wide spectrum of species and cell types, and to identify Have been viewed as gene delivery vectors due to their ability to be packaged in cell lines. Other viruses, such as adenovirus, herpes simplex virus (HSV), cytomegalovirus (CMV), and adeno-associated virus (AAV) (see, for example, US Pat. No. 5,139,941; herein incorporated by reference) The virus described in i)) can also be engineered to act as a vector for gene transfer.

Although some viruses that can accept exogenous genetic material are limited in the number of nucleotides they can accommodate, and in the range of cells in which they infect, these viruses have successfully modified gene expression. It has been proven to bring. However, adenoviruses do not integrate their genetic products into the host genome and therefore do not require host replication for gene expression. This makes them ideally suited for rapid gene expression, efficient gene expression, heterologous gene expression. Techniques for preparing replication-defective infectious viruses are well known in the art.

[0512] In certain further embodiments, the gene therapy vector is HSV. HS
Factors that make V an attractive vector are the size and organization of the genome. HS
Because V is large, integrating multiple genes or expression cassettes is less of a problem than other smaller viral systems. In addition, the availability of various virus control sequences with varying performance (eg, temporal, intensity) allows for controlling expression to a greater extent than in other systems. It would also be useful for the virus to have a relatively small number of spliced messages to further facilitate genetic manipulation. HSV is also relatively easy to manipulate and can be grown to high titers.

Of course, when using a viral delivery system, unwanted contaminants (eg,
Virions are purified sufficiently to be essentially free of incomplete interfering virions or endotoxins, and other pyrogens, so that they are not inconvenient in cells, animals, or individuals receiving the vector construct. It is desired not to cause any reaction. A preferred means of purifying the vector is to use a buoyant density gradient (
For example, cesium chloride gradient centrifugation).

C8. Antibody Conjugates Can VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies be conjugated to an anti-cellular agent or negative to prepare an “immunotoxin”;
Alternatively, it can be operably associated with a component that can directly or indirectly stimulate coagulation, thereby forming a "coag ligand". In a coag ligand, the antibody can be directly linked to a direct or indirect clotting factor, or can be linked to a second binding region that binds and then releases the direct or indirect clotting factor. The "second binding region" approach generally uses a coagulant-binding antibody as the second binding region, thereby producing a bispecific antibody construct. The preparation and use of bispecific antibodies is generally well known in the art and is further disclosed herein.

In preparing immunotoxins, coag ligands and bispecific antibodies, recombinant expression can be used. The nucleic acid sequence encoding the selected antibody is linked in-frame to the nucleic acid sequence encoding the selected toxin, coagulant, or second binding region, creating an expression unit or vector. I do. Recombinant expression results in translation of a new nucleic acid, yielding the desired protein product. Although a nucleic acid encoding the antibody is used rather than a protein binding ligand, the recombination approach is essentially the same as the one described herein above.

Returning to the conjugate technology, the preparation of immunotoxins is generally well known in the art. However, certain advantages, both in the preparation of immunotoxins and in their purification for subsequent clinical administration, can be achieved through the application of certain preferred techniques. For example, while IgG-based immunotoxins typically show better binding capacity and slower blood clearance than their Fab 'counterparts, Fab' fragment-based immunotoxins generally
Shows better tissue penetration capacity when compared to G-based immunotoxins.

In addition, although many types of disulfide bond-containing linkers are known that can be used successfully to conjugate a toxin moiety to a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody, certain linkers are generally Preferred over other linkers based on differences in pharmacological characteristics and capabilities. For example, linkers containing disulfide bonds that are sterically "hindered" are preferred due to their greater stability in vivo, thereby preventing release of the toxin moiety prior to attachment at the site of action.

A wide variety of negatives (toxins from plants, toxins from fungi and toxins from bacteria, such as ricin A chain or deglycosylated A, which can be conjugated to VEGFR2 blocking anti-VEGF antibodies or The cross-linking of the toxin A chain to the targeting agent in certain cases requires a cross-linking agent exhibiting a disulfide function, the reason for which is unknown, but once this factor It seems that due to the "delivery" of this toxin to target cells, the need for specific toxin moieties that can be readily released from the targeting agent.

Each type of crosslinking agent, as well as the manner in which crosslinking is performed, tends to vary the pharmacodynamics of the resulting conjugate. Ultimately, if a releasable toxin is intended,
It is desirable to have a conjugate that remains intact under the conditions found throughout the body except for the intended site of action (preferably the conjugate has good "release" characteristics). Thus, the particular crosslinking scheme, including the particular crosslinking reagent used and the structure to be crosslinked, is somewhat important.

Depending on the particular toxin compound used as part of the fusion protein, it may be necessary to provide antibodies and toxin compounds that operably bind to the targeting agent. Proteolytic cleavage within the loop then results in a heterodimeric polypeptide, wherein the antibody and toxin compound are linked only by one disulfide bond. An example of such a toxin is ricin A chain toxin.

When certain other toxin compounds are utilized, the non-cleavable peptide spacer is
An EGFR2-blocking anti-VEGF antibody or 2C3-based antibody and a fusion protein toxin compound are provided for operably linking. Toxins that can be used in combination with a non-cleavable peptide spacer are themselves toxins that can be converted to a cytotoxic disulfide-linked form by proteolytic cleavage. An example of such a toxin compound is a Pseudonomas exotoxin compound.

[0522] Targeted antigens, such as those desired to target chemotherapeutic agents (eg, anti-tumor drugs, other cytokines, antimetabolites, alkylating agents, hormones, etc.), may be more efficient than immunotoxins. There may be situations such as when not internalized by a path consistent with addiction. A variety of chemotherapeutic and other pharmacological agents are now successfully conjugated to antibodies and have been shown to function pharmacologically. Exemplary anti-tumor agents being investigated are doxorubicin, daunomycin,
Including methotrexate, vinblastine, and various other factors. In addition, other factors (eg, neocarzinostatin)
, Macromycin, trenimon
And portions of α-amanitin have been described.

When a clotting factor is used in combination with the present invention, any covalent attachment to the antibody should be made at a site other than its functionally clotting site. Thus, the composition is "linked" in any operable manner, such that each region performs its intended function without significant obstruction. Therefore, this antibody
It binds to VEGF and coagulation factors promote clot formation.

C9. Biochemical Crosslinking Agents In addition to the general information provided above, VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies may be used as anti-cellular agents using certain preferred biochemical crosslinking agents. Or it can be negatively conjugated. Cross-linking reagents are used to form molecular cross-links that link together functional groups of two different molecules. To link two different proteins in a stepwise manner, heterobifunctional crosslinkers that eliminate unwanted homopolymer formation can be used. Exemplary heterobifunctional crosslinkers are listed in Table B1
Referred to.

[0525]

[Table 2]

A heterobifunctional crosslinker has two reactive groups, one generally a primary amine group (
(E.g., N-hydroxysuccinimide) and the other generally contains a thiol group (e.g., reacting with pyridyl disulfide, maleimide, halogen, etc.). Through a primary amine reactive group, the crosslinker can react with a lysine residue of a protein (eg, a selected antibody or fragment) and is already associated with the first protein through the thiol reactive group. Crosslinking agents include cysteine residues (free sulfhydryl groups) on other proteins (eg, clotting drugs)
Reacts with.

Thus, the compositions generally have or are derivatized to have functional groups available for crosslinking purposes. This requirement is not considered to be limiting in that a wide variety of groups can be used in this manner. For example, primary or secondary amine groups, hydrazide or hydrazine groups, carboxyl groups, alcohol groups, phosphate groups, or alkylating groups can be used for conjugation and crosslinking.

The spacer arm between the two reactive groups of the crosslinker can have various lengths and chemical compositions. Longer spacer arms allow better flexibility of the conjugate component, while some specific components in the crosslink (eg, benzene groups)
May provide additional stability to the reactive group, or increased resistance of chemical bonds to various aspects of the action (eg, disulfide bonds resistant to reducing agents). The use of a peptide spacer (eg, L-Leu-L-Ala-L-Leu-L-Ala) is also contemplated.

Preferably, a crosslinking agent having a reasonable stability in blood is used. Can be successfully used to conjugate targeting agents and toxin or clotting factors,
Many types of linkers containing disulfide bonds are known. A linker containing a sterically hindered disulfide bond may prove to produce greater stability in vivo, which prevents release of the factor prior to binding at the site of action. Thus, these linkers are one preferred group of linking agents.

One of the most preferred cross-linking reagents for use in immunotoxins is SMP
T. SMPT is a bifunctional crosslinker that contains disulfide bonds that are “sterically hindered” by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond protects the bond from attack by thiolate anions (such as glutathione, which may be present in tissues and blood), thereby removing the conjugate prior to delivery of the bound drug to the tumor site. It is thought to provide a useful function to prevent coupling. It is contemplated that SMPT agents may also be used in combination with the bispecific ligands of the present invention.

[0531] Like many other known cross-linking reagents, SMPT cross-linking reagents are functional groups (eg,
Provides the ability to crosslink the cysteine SH or primary amines (eg, the lysine ε-amino group). Another possible type of crosslinker is a cleavable disulfide bond (eg, sulfosuccinimidyl-2- (p-azidosalicylamido) ethyl-1,
3'-dithiopropionate), including heterobifunctional photosensitive phenyl azides. The N-hydroxy-succinimidyl group reacts with a primary amino group,
And phenyl azide (during photolysis) reacts non-selectively with any amino acid residue.

In addition to hindered crosslinkers, unhindered linkers can also be used according to the present specification. Other useful crosslinkers that do not appear to contain or produce protected disulfides include SATA, SPDP and 2-iminothiolene. The use of such crosslinking agents is well understood in the art.

Once conjugated, the conjugate is separated from unconjugated targeting and unconjugated therapeutic agents, and from other contaminants. Numerous purification techniques are available for use that provide conjugates of sufficient purity to render the conjugate clinically useful. Purification methods based on size separation (eg, gel filtration, gel permeation, or liquid high performance chromatography) are generally most useful. Other chromatography techniques such as Blue-Sepharose separation can also be used.

C10. Biologically Releasable Linkers The optional binding moiety has reasonable stability in the blood and substantially prevents the attached drug from being targeted before targeting it to a disease or tumor site. Although it is preferred to prevent rapid release, in certain aspects, the use of biologically releasable bonds and / or selectively cleavable spacers or linkers is contemplated. "Biologically releasable bonds" and "selectively cleavable spacers or linkers" still have reasonable stability in circulation.

Thus, a VEGFR-2 blocking anti-VEGF antibody of the invention (eg, a 2C3-like antibody) can be conjugated to one or more therapeutic agents via a biologically releasable linkage. Sc
Although Fv fragments are preferred in certain embodiments, VEGFR2
Any form of blocking anti-VEGF antibody, or "target antibody or drug", can be used, including intact antibodies.

A “biologically releasable bond” or “selectively hydrolysable bond” is any bond that is releasable, cleavable, or hydrolyzable under certain conditions, either exclusively or preferentially. Including binding. This is disclosed in US Pat. Nos. 5,474,765 and 5,474,765.
762,918, each of which is specifically incorporated herein by reference.
And disulfide and trisulfide bonds, as well as acid-labeled bonds.

The use of an acid-sensitive spacer for attachment of a therapeutic or therapeutic agent to an antibody of the invention is specifically contemplated. In such embodiments, the therapeutic agent or drug is released within the acid compartment within the cell. Acid-sensitive release can occur extracellularly,
After a particular label, it is preferably intended to occur against the tumor site. Certain currently preferred examples include 2C3-like antibodies conjugated to colchicine or doxorubicin via an acid-sensitive spacer. Attachment through the carbohydrate moiety of the antibody is also contemplated. In such embodiments, the therapeutic agent or drug is released within the acidic compartment within the cell.

The targeted anti-VEGF antibody can also be directed to introduce a functional group that allows for attachment of the therapeutic via a biologically releasable linkage. Thus, a target antibody can be induced to introduce a side chain terminator at a hydrazide, hydrazine, primary or secondary amine group. Therapeutic agents can be conjugated via a Schiff base bond, a hydrazone or acyl hydrazone bond or a hydrazide linker (US Pat. Nos. 5,474,765 and 5,762,918).
, Each of which is specifically incorporated herein by reference).

US Pat. Nos. 5,474,765 and 5,762,918, each of which is incorporated herein by reference.
Targeted anti-VE, as described in US Pat.
The GF antibody may comprise one or more biologically releasable linkages (peptide bonds,
(Including esters, amides, phosphodiesters and glycosides).

[0540] A preferred aspect of the present invention is that it is preferentially located within a disease site, particularly a tumor environment,
It relates to the use of a peptide linker comprising at least a first cleavage site for peptidases and / or proteinases. Thus, antibody-mediated delivery of an attached therapeutic agent results in specific cleavage at the disease site or within the tumor environment, resulting in specific release of the active agent. Certain peptide linkers contain cleavage sites that are recognized by one or more enzymes for reassembly.

Urokinase, pro-urokinase, plasmin, plasminogen, TGFβ
Particularly preferred are polypeptide linkers that comprise a cleavage site for staphylokinase, thrombin, factor IXa, factor Xa or a metalloproteinase (eg, interstitial collagenase, gelatinase, or stromelysin). U.S. Patent No. 6,004,555, U.S. Patent No. 5,877,289 and U.S. Application No. 08 / 482,369 (issued October 20, 1998, paid) provide further description and targeted drug-therapeutic agents. For the purpose of enabling methods of making and using constructs, including biologically releasable bonds and selectively cleavable linkers and peptides, are specifically incorporated herein by reference. You. In particular,
U.S. Patent No. 5,877,289, issued March 2, 1999, provides further description and targeted drug-therapeutic agent construction (urokinase, plasmin, thrombin,
Methods of making and using factor IXa, factor Xa or metalloproteinases (eg, including a selectively cleavable peptide linker that is cleaved in the tumor environment by interstitial collagenase, gelatinase, or stromelysin) Is specifically incorporated herein by reference for the purpose of enabling

Presently preferred selectively cleavable peptide linkers are cleavages for plasmin or metalloproteinases (also known as “matrix metalloproteases” or “MMPs”), such as interstitial collagenase, gelatinase or stromelysin A linker containing a site. Additional peptide linkers that can be used in connection with the present invention include, for example, the linkers listed in Table B2.

[0543]

[Table 3] C11. Bispecific Antibodies Bispecific antibodies are particularly useful in the coag ligands of the invention and in the context of combined anti-angiogenesis. However, bispecific antibodies generally have one arm binding to VEGF (substantially the same epitope as 2C3 if necessary) and the bispecific antibody binding to a therapeutic (generally antigen binding). As long as it binds (at a site different from the site). Bispecific antibodies that bind to both PS and PE are also
Can be used.

Generally, the preparation of bispecific antibodies is also well known in the art. One
The method comprises, on the one hand, an antibody having specificity for a target antigen, on the other hand,
And (2) preparing the flocculants separately. F (ab'γ) of pepsin _Two Fragments were prepared from the two selected antibodies, followed by reduction of each, separately
Fab '[gamma]_SHProvide a fragment. Next, two parts to be coupled
The SH group in one of the two groups is replaced with a crosslinking agent such as o-phenylenedimaleimide.
To provide a free maleimide group on one partner. Next
Is linked to the other by means of a thioether bond and the desired F (
ab '[gamma])_TwoHeteroconjugates can be produced. Using SPDP or Protein A
Other techniques for performing cross-linking or preparing trispecific constructs are known.

[0545] Another method of generating bispecific antibodies is to fuse two hybridomas to form a quadroma. As used herein, the term "quadroma" is used to describe a fusion product of two B cell hybridomas. Here, cells that fuse the two antibody-producing hybridomas using standard techniques, produce daughter cells, and maintain expression of both sets of clonogenic immunoglobulin genes are then selected. .

A preferred method of producing quadromas involves the selection of a mutant that lacks at least one enzyme of the parent hybridoma. This first mutant hybridoma cell line is then fused to cells of a second hybridoma that has been exposed to lethal exposure to, for example, iodoacetamide, which prevents its continued survival. Cell fusion involves the rescue of a first hybridoma and the rescue of a second hybridoma through fusion with the first hybridoma by obtaining the gene for the enzyme deletion from the lethally processed hybridoma. Make it possible. Fusion of immunoglobulins of the same isotype but of different subclasses is preferred, but not required. If alternative assays exist for the isolation of the preferred quadromas, mixed subclass antibodies can be used.

More particularly, one method of quadroma development and screening involves obtaining a hybridoma strain that secretes a first selected MAb, and converting the hybridoma to an essential metabolic enzyme (hypoxanthine-guanine phospholipid). Ribosyltransferase (HGPRT)). To obtain deletion mutants of hybridomas, cells were grown at increasing concentrations of 8-azaguanine (1 ×
(10 ⁻⁷ M to 1 × 10 ⁻⁵ M). Mutants were subcloned by limiting dilution and their hypoxanthine / aminopterin / thymidine (
HAT) Test for sensitivity. This culture medium is, for example, 10% FCS,
It may consist of DMEM supplemented with 2 mM L-glutamine and 1 mM penicillin-streptomycin.

A quadroma is produced by standard cell fusion techniques using a complementary hybridoma cell line that produces a second desired MAb. In short, 4.5 × 1
0 ⁷ a first HAT-sensitive cells of, 2.8 × 10 ⁷ second cells HAT resistance (irreversible biochemical inhibitor is a lethal dose of iodoacetamide (5 mM in phosphate buffered saline) For 30 minutes prior to fusion on ice). Cell fusion is induced using polyethylene glycol (PEG) and the cells are plated in 96-well microculture plates. Quadromas are selected using HAT containing media. Bispecific antibody-containing cultures, for example,
Identification is performed using solid-phase isotype-specific ELISA and isotype-specific immunofluorescent staining.

To identify bispecific antibodies, in one identification embodiment, the wells of a microtiter plate (Falcon, Becton Dickinson)
Labware) is coated with a reagent that specifically interacts with one of the parent hybridoma antibodies and has no cross-reactivity with both antibodies. The plate is washed, blocked, and the supernatant to be tested (SN) is added to each well. The plate is incubated for 2 hours at room temperature, the supernatant is discarded, the plate is washed and the diluted alkaline phosphatase-anti-antibody conjugate is added for 2 hours at room temperature. The plate is washed and a phosphatase substrate (e.g., p-nitrophenyl phosphate (Sigma, St. Louis)) is added to each well. The plate was incubated, the reaction was stopped by adding 3N NaOH to each well, and E
The OD ₄₁₀ value is measured using a LISA reader.

In another identification embodiment, a microtiter plate pre-treated with poly-L-lysine is used to bind one of the target cells to each well and then immobilize the cells (eg, 1% glutaraldehyde), and the bispecific antibody is tested for its ability to bind to intact cells. Furthermore, FA
CS, immunofluorescent staining, idiotype-specific antibodies, antigen binding competition assays, and other methods common in the field of antibody characterization can be used in combination with the methods of the invention to identify preferred quadromas.

Following quadroma isolation, the bispecific antibody is purified from other cellular products. This purification can be achieved by various protein isolation procedures known to those skilled in the art of immunoglobulin purification. Means for preparing and characterizing antibodies are well known in the art (eg, Antibodies: AL).
laboratory Manual, 1988).

For example, the supernatant of the selected quadroma is passed through a protein A sepharose column or a protein G sepharose column to bind the IgG (depending on the isotype). The bound antibody is then eluted, for example, using a pH 5.0 citrate buffer. The eluted fraction containing BsAb is dialyzed against an isotonic buffer. Alternatively, the eluate is also passed over an anti-immunoglobulin sepharose column. Next, the BsAb is eluted using 3.5M magnesium chloride. The BsAbs purified in this manner are then tested for binding activity, for example, by the target cell isotype-specific ELISA and immunofluorescent staining assays described above.

The purified BsAb and the parent antibody can also be characterized and isolated by SDS-PAGE electrophoresis followed by silver or Coomassie staining. This procedure is possible if one of the parent antibodies has a higher molecular weight than the other antibody and the BsAb band migrates between the two parent antibodies. The reduction of the sample
The presence of two apparently different molecular weight heavy chains is confirmed.

D. Pharmaceutical Compositions The pharmaceutical compositions of the invention generally comprise at least a first VEGFR2 blocking anti-VE.
An effective amount of a GF antibody or 2C3-based antibody (dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium). Combination treatments are also contemplated,
And the same type of underlying pharmaceutical composition may be used for both single and combination medicines.

The phrase “pharmaceutically-acceptable or pharmacologically-acceptable,” optionally, results in a deleterious, allergic or other adverse reaction when administered to an animal or human. Refers to the entire molecule or composition. Veterinary use, equally encompassed by the present invention, and "pharmaceutically acceptable" formulations include those for both clinical and / or veterinary use.

As used herein, “pharmaceutically acceptable carrier” includes:
Any and all solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents and adsorption delaying agents and the like are included. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. Supplementary active ingredients can also be incorporated into the compositions.

A “unit dose” formulation includes a dose or sub-dose of the administered ingredient adapted for a particular timely delivery. For example, an exemplary "unit dose" formulation is a formulation that includes a daily dose or daily dose unit or sub-dose or a weekly dose or weekly dose unit or weekly sub-dose and the like.

D1. Injectable Formulations VEGFR2-blocking anti-VEGF antibody-based or 2C3-based antibodies or immunoconjugates of the invention are most often administered parenterally (eg, orally, intravenously, intramuscularly, subcutaneously, transdermally). It is formulated for injection via the skin or for peristaltic administration and other routes including direct instillation into the tumor or disease site (intracavitary administration). The preparation of an aqueous composition that contains a therapeutic-targeting agent construct as an active ingredient will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either in aqueous solutions or suspensions; for addition to liquids prior to injection, to prepare solutions or suspensions. Solid forms suitable for use may also be prepared; and the preparation may also be emulsified.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or suspensions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile injectable solutions or injectable suspensions. Sterile powders for the extemporaneous preparation of In all cases, the form should be sterile and should be fluid to the extent that injectability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.

A VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate composition can be formulated in a neutral or salt form in a sterile aqueous composition. Solutions as a free base, or as a pharmacologically acceptable salt, can be added to a surfactant (eg,
(Hydroxypropylcellulose) in water appropriately mixed. Pharmaceutically acceptable salts include acid addition salts (formed using the free amino groups of proteins), and inorganic acids (eg, hydrochloric or phosphoric acid) or organic acids (eg, acetic acid, trifluoroacetic acid) Oxalic acid, tartaric acid, mandelic acid) and the like. Salts formed with the free carboxy groups also
It can be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron (III) hydroxide and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.

[0566] Suitable carriers include, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like).
And suitable mixtures thereof, as well as solvents and dispersion media, including vegetable oils. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Proper fluidity can be maintained, for example, by the use of a coating (eg, lecithin), by the maintenance of the required particle size in the case of dispersion, and / or by the use of surfactants.

Under ordinary conditions of storage and use, all such preparations should contain a preservative to prevent the growth of microorganisms. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of delaying agents, such as aluminum monostearate and gelatin.

Upon or prior to formulation, the VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate may be extensively dialyzed to remove unwanted small molecule substances and / or It should be lyophilized for easier formulation into the desired vehicle. Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various other ingredients enumerated above, if necessary, followed by sterile filtration. Is done. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of a sterile injectable solution, the preferred method of preparation is to dry and freeze-dry to produce a powder of the active ingredient and any further desired ingredients from that solution previously sterile filtered. Dry technology.

[0566] Suitable pharmaceutical compositions according to the invention generally comprise an amount of a VEGFR2-blocking anti-VEGF mixed with an acceptable pharmaceutical diluent or excipient (eg, a sterile aqueous solution).
Including the F antibody or 2C3-based antibody or immunoconjugate, yields a range of final concentrations, depending on the intended use. The technology of preparation is based on Remington's
Pharmaceutical Sciences, 16th Edition, Mack Pu
It is generally well known in the art, as exemplified by the Brushing Company, 1980, which is incorporated herein by reference. It should be understood that endotoxin contamination should be kept to a safe level to a minimum (eg, less than 0.5 ng / mg protein). In addition, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologics standards. Upon formulation, antibody or immunoconjugate solutions will be administered in a manner compatible with the dosage formulation, and in such therapeutically effective amount.

D2. Sustained Release Formulations Formulations of VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugate solutions are readily available in a variety of dosage forms (eg, in the form of injectable solutions described above). Administered, but in other pharmaceutically acceptable forms (eg, tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomes Forms) are also contemplated. The type of dosage form is adapted to the disease or disorder to be treated.

Pharmaceutical “slow release” capsules or “slow release” compositions or preparations can be used and are generally applicable. Sustained-release formulations are generally designed to produce a constant drug level over an extended period of time, and
It can be used to deliver blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugates.

Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices can be formed into shaped articles (eg, films or microparticles). Capsule). Examples of sustained release matrices include: polyester; hydrogel (eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)); polylactide (eg, US Pat. No. 3,773,919). ); Copolymers of L-glutamic acid and γ-ethyl L-glutamate; non-degradable ethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers (eg, Lupron De)
potTM (injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate)); and poly-D-(-)-3-hydroxybutyric acid.

Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, and certain hydrogels release proteins for shorter time periods. If the encapsulated antibodies remain in the body for an extended period of time, they may denature or aggregate as a result of exposure to moisture at 37 ° C., thus reducing biological activity and / or altering immunogenicity. Rational strategies are available for stabilization depending on the mechanism involved. For example, if the aggregation mechanism involves intermolecular SS bond formation via thio-disulfide interconversion, stabilization may include modification of sulfhydryl residues, lyophilization from acidic solutions, control of water content, appropriate This is achieved through the use of additives, the development of specific polymer matrix compositions, and the like.

D3. Liposomes and Nanocapsules In certain embodiments, liposomes and / or nanocapsules also
It can be used with EGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugates. The formulation and use of liposomes is generally known to those skilled in the art, as summarized below.

[0571] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form a multilayered, concentric bilayer vehicle (also referred to as a multilayered vehicle (MLV)). MLVs generally have a diameter between 25 nm and 4 μm. Sonication of the MLV produces a small monolayer vehicle (SUV) ranging from 200-500 ° in diameter containing an aqueous solution in the center.

[0572] Phospholipids, when dispersed in water, can form a variety of structures other than liposomes, depending on the molar ratio of lipid to water. At low ratios, liposomes are the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes can exhibit low permeability to ions and polar substances, but at elevated temperatures undergo a phase transition that significantly changes their permeability. Phase transition involves the change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less ordered structure, known as the fluid state. This occurs at characteristic phase transition temperatures and increases the permeability of ions, sugars and drugs.

Liposomes interact with cells through four different mechanisms: endocytosis by phagocytes of the reticuloendothelial system, such as macrophages and neutrophils; nonspecific weak hydrophobic forces, or static Adsorption to the cell surface, either by electrical forces or by specific interaction with cell surface components; liposome lipid bilayers attach to the plasma membrane with simultaneous release of liposome contents into the cytoplasm Fusion with the plasma cell membrane by insertion; and the transfer of liposome lipids to the cell membrane or to the intracellular (organelle) membrane without any association of the liposome contents, or vice versa. Although more than one mechanism can operate simultaneously, changing the liposome formulation can change the mechanism that operates.

The nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to polymeric overload in cells,
Such ultrafine particles (about 0.1 μm in size) should be designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily made.

D4. Ophthalmic Formulations Many diseases with vasogenic components involve the eye. For example, diseases relating to corneal neovascularization that can be treated according to the present invention include diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and post-lens fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency , Contact lens overwear, atopic keratitis, upper limbal keratitis, pterygium keratitis syndrome, Sjogren, acne rosacea, frychtenosis (phy
syphilis), syphilis, mycobacterial infection, lipid degeneration, chemical ablation, microbial ulcer, fungal ulcer, herpes simplex infection, shingles infection, kaposi's sarcoma, Mohren's ulcer, terien marginal degeneration, marginal keratolysis, trauma, rheumatoid Arteritis, systemic lupus, polyarteritis, Wegener's sarcoma, scleritis, Stevens-Johnson disease, periphigoid radial keratotomy, and corneal graft rejection.

Diseases relating to retinal / choroidal neovascularization that can be treated according to the present invention include diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, elastofibrous pseudoxanthoma, Paget's disease, venous atresia , Arterial closure, carotid occlusive disease, chronic uveitis /
Vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, diseases causing Eiles disease, Becht's disease, retinitis or choroiditis, putative ocular histoplasmosis, best disease, myopia, optic disc. Deficiency, stargart disease, pars planiti
s), chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, trauma and post-laser complications.

Other diseases that may be treated in accordance with the present invention include flushing skin (corneal neovascularization) and hyperproliferation of vascular connective or fibrous tissue (proliferative vitreous, with or without diabetes) (Including but not limited to, all forms of retinopathy).

Thus, VEGFR2-blocking anti-VEGF antibody-based antibodies of the invention and 2C3
The base antibody as well as the immunoconjugate can be advantageously used in the preparation of a pharmaceutical composition suitable for use as an ophthalmic solution, wherein the pharmaceutical composition is administered intravitreally and / or intracamerally. A composition for VEGFR2 blocking compositions of the invention for the treatment of the aforementioned disorders or other disorders,
Either EGF antibody compositions or 2C3-based antibody compositions are compatible with conventional pharmaceutical practices (eg, “Remington's Pharmaceutical S”).
sciences, 15th Edition, pp. 1488-1501 (Mack Publish
ing Co. , Easton, PA) in the form of an ophthalmic preparation, which is administered to the subject's eye in need of treatment.

The ophthalmic preparations may be present in a pharmaceutically acceptable solution, suspension or ointment from about 0.01 to about 10%.
VEGFR2 blocking anti-VEGF antibody or 2C3-based antibody at a concentration of 1% by weight, preferably about 0.05 to about 0.5% by weight. Some variation in concentration will occur depending on the particular compound used, the condition of the subject being treated, and the like, and the treating practitioner will determine the most appropriate concentration for the individual subject. The ophthalmic preparation is preferably in the form of a sterile aqueous solution, and if desired, further ingredients such as preservatives, buffers, elasticizers, antioxidants and stabilizers, nonionic wetting or water purifying agents, Includes viscosity enhancers.

Suitable preservatives for use in such solutions include benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal, and the like. Suitable buffers include between about pH 6 and pH 8, preferably about p
Boric acid, sodium and potassium bicarbonate, sodium and potassium borate, sodium and potassium carbonate, sodium acetate, sodium biphosphate, and the like in amounts sufficient to maintain a pH between H7 and pH 7.5. Is mentioned. Suitable elasticizers are Dextran 40, Dextran 70
, Dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, etc., and the sodium chloride equivalent of the ophthalmic solution is in the range of 0.9 ± 0.2%.

Suitable antioxidants and stabilizers include sodium bisulfite, sodium bisulfite, sodium thiosulfite, thiourea and the like. Suitable humectants and water purifiers include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity increasing agents include dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose, and the like. Ophthalmic preparations
It is administered topically to the subject's eye in need of treatment, eg, in the form of drops, by conventional means, or by immersing the eye in an ophthalmic solution.

D5. Topical Formulations In a broad sense, formulations for topical administration include those for delivery via the mouth (buccal) and through the skin. "Topical delivery systems" also include transdermal patches containing the ingredient to be administered. Delivery through the skin can be further achieved, if desired, by ion implantation or electrical delivery.

Formulations suitable for topical administration in the mouth include flavored base ingredient,
A lozenge usually containing sucrose and acacia or tragacanth; an active ingredient in an inert base material (eg, gelatin and glycerin) or a pill containing sucrose and acacia; and a gargle containing the ingredient to be administered in a suitable liquid carrier. Medicines.

[0591] Suitable formulations for topical administration to the skin include ointments, creams, gels and plasters, including those administered with a pharmaceutically acceptable carrier.
Formulations of VEGFR2-blocking anti-VEGF or 2C3-based antibodies for topical use (eg, in creams, ointments and gels) include preparations of fatty bases or water-soluble ointments, as are well known in the art. Is mentioned. For example, these compositions may include vegetable oils, animal fats, and more preferably, semi-solid hydrocarbons derived from petroleum. Specific ingredients used are white ointment, yellow ointment, cetyl ester wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, glycerin starch, white wax, yellow wax, lanolin, anhydrous lanolin and monostearin Glyceryl acid may be mentioned. A variety of water-soluble ointments may be used, including glycol ethers and derivatives, polyethylene glycol, polyoxyl 40 stearate, and polysorbate.

Formulations for rectal administration may be presented as a suppository with a suitable base including, for example, cocoa butter or a salicylate. Suitable formulations for nitrogen administration include pessaries, tampons, creams, gels, plasters, wraps, or spray formulations containing additional active ingredients (eg, carriers known in the art to be suitable). Can be given as

D6. Nasal Formulations Local delivery via the nasal and respiratory routes is intended to treat various conditions. These delivery routes are also suitable for delivering drugs to the systemic circulation. Thus, the formulation of the active ingredient in a suitable carrier for nasal administration is
Included within the present invention are, for example, nasal solutions, sprays, aerosols and inhaled antigens. Where the carrier is an individual, the formulation may, for example, include a coarse powder having a particle size in the range of 20 to 500 microns, e.g., a rapid powder passing through the nasal passage from a powder container maintained in close proximity to the nose. Administered by inhalation.

[0591] Suitable formulations where the carrier is a liquid are useful for nasal administration. Nasal solutions are aqueous solutions usually designed to be administered to the nasal route by drops or sprays, and they are similar in many aspects to nasal secretions, maintaining normal ciliary action It is prepared as follows. Thus, nasal aqueous solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 6.5. In addition, if necessary, antimicrobial preservatives, similar to those used in ophthalmic preparations, and suitable drug stabilizers may be included in the formulation. Various commercial nasal preparations are known and include, for example, antibiotics and antihistamines, and are used for asthma prevention.

Inhalants and inhalants are pharmaceutical preparations designed for delivery of drugs or compounds to the respiratory tree of a patient. Steam or fog is administered and reaches the active area. This route can also be used to deliver drugs to the systemic circulation. Inhalants may be administered by the nasal or oral respiratory route. Administration of the inhalant solution is effective only if the droplets are of good quality and uniform in size, so that the mist reaches the bronchioles.

Another group of products (also known as inhalants, and sometimes referred to as inhalants) are high quality powdered or liquid drugs (eg, pharmaceuticals) that are delivered to the respiratory tract by use of specific delivery systems. Aerosol) to maintain a solution or suspension of the drug in a liquid gas propellant. When released through a suitable valve and mouth adapter, a measured dose of the inhaled drug is advanced into the patient's respiratory tract. Particle size is of primary importance in the administration of this type of preparation. The optimal particle size for penetration into the lung cavity has been reported to be on the order of 0.5-7 μm. Good quality mist is produced by pressurized aerosols, whereby the advantages of their use are:
Be considered.

E. Therapeutic Kits The present invention also provides VEGFR2 blocking anti-V for use in the treatment methods of the invention.
There is provided a therapeutic kit comprising an EGF antibody or a 2C3-based antibody or an immunoconjugate. Such a kit generally contains, in suitable container means, a pharmaceutically acceptable formulation of at least one VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate. The kit also includes other pharmaceutically acceptable formulations for either diagnostic / imaging or combination therapy. For example, such kits may include a range of chemotherapeutic or radiotherapeutic agents; anti-angiogenic agents; anti-tumor cell antibodies; and / or anti-tumor vasculature or anti-tumor stroma immunotoxins or coag ligands. May be included.

The kit comprises a single container containing a VEGFR2-blocked anti-VEGF antibody or 2C3-based antibody or immunoconjugate with or without any additional components (
Container means) or a separate container for each desired drug. When a combination therapeutic is provided, a single solution may be pre-mixed, either in molar equivalent combination or in one component in excess of the other. Alternatively, each VEGFR2 blocking anti-VEGF antibody or 2C in the kit
The tri-based antibody or immunoconjugate and other anti-cancer agent components can be separately maintained in different containers prior to administration to a patient.

When the components of the kit are provided in one or more liquids, the liquid is preferably an aqueous solution, especially a sterile aqueous solution. However, the components of this kit
It may be provided in a dry powder. When the reagents or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be provided in another container.

The kit's container will generally include at least one vial, test tube, flask, bottle, syringe, or other container means in which the VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate and Any other desired agent can be placed, preferably in a suitable aliquot. Where separate components are included, the kit will also generally include a second vial or other container within which the components will be placed to allow administration at separately designed doses. The kit may also include a second / third container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.

The kit also provides a means for administering a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate to an animal or patient (eg, one or more needles or syringes, or eye drops, pipettes, or other By such means, the formulation may be injected into an animal or applied to a diseased area of the body. The kits of the invention also typically include means (eg, injection molded or blow molded plastic containers) containing vials and the like, and other components in close restraint for commercial sale, and include At this point, the desired vials and other equipment are placed and held.

F. Anti-Angiogenic Therapies The present invention can be used, for example, to treat animals and patients with abnormal angiogenesis that contribute to various diseases and disorders. The most prevalent and / or clinically important of these, apart from the field of cancer treatment, include:
Joint deformity, rheumatoid arthritis, psoriasis, atherosclerosis, diabetic retinopathy, age-related macular degeneration, Graves' disease, vascular restenosis (including the following restenosis: angioplasty, arteriovenous insufficiency ( AVM), meningioma, hemangiomas and neovascular glaucoma). Other potential targets for intervention include: hemangiofibromas, atherosclerotic plaques, corneal transplant neovascularization, hemophilic joints, hypertrophic scars, Ausler-Weber syndrome, Purulent granulomas Post-lens fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis, various other inflammatory diseases and disorders, and endometriosis. Additional diseases and disorders treatable by the present invention, as well as a unified basis for such angiogenic disorders, are described below.

One disease associated with angiogenesis is rheumatoid arthritis, where the blood vessels in the synovial lining of the joint undergo angiogenesis. In addition to forming new vascular networks, endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. Factors associated with angiogenesis may actively contribute to and maintain the chronic inflammatory state of rheumatoid arthritis. Factors associated with angiogenesis also have a role in osteoarthritis and contribute to joint destruction.

[0597] Harada et al. (1998, specifically incorporated herein by reference) state that VEGF has been implicated in the pathogenesis of rheumatoid arthritis, and furthermore, that measurement of serum levels of VEGF has been shown to be useful in rheumatoid arthritis. Has been shown to be a useful non-invasive method for monitoring the disease activity of s. This supports the therapeutic and diagnostic uses of the present invention in connection with rheumatoid arthritis.

[0598] Nagashima et al. (1999, specifically incorporated herein by reference) describe the inhibitory effect of anti-rheumatic drugs on VEGF in cultured rheumatoid synovial cells. VEGF is constitutively expressed in the synovium of rheumatoid arthritis. Known antirheumatic drugs (Bucilamine (BUC)) have been shown to include within its mechanism of action the inhibition of VEGF production by synovial cells. Thus, the anti-rheumatic effect of BUC is mediated by inhibition of angiogenesis and synovial proliferation in the rheumatoid synovium via inhibition of VEGF production by synovial cells. The use of the present invention as an anti-rheumatic treatment is supported by the VEGF inhibitory effects of this existing therapeutic.

Another example of a disease mediated by angiogenesis is an ocular neovascular disease. The disease is characterized by the invasion of new blood vessels into the structure of the eye, such as the retina or cornea. It is the most common cause of blindness and is associated with about 20 eye diseases. In age-related macular degeneration, a related visual problem is caused by the ingrowth of choroid capillaries via a defect in Bruch's membrane with proliferation of vascular tissue beneath the retinal pigment epithelium. Angiogenic lesions are also associated with diabetic retinopathy, retinopathy of prematurity, corneal transplant rejection, neovascular glaucoma, and post lens fibroplasia.

Other diseases associated with corneal neovascularization include, but are not limited to: pandemic keratoconjunctivitis, vitamin A deficiency, contact lens overload (o
verwear), atrophic keratitis, upper limbal keratitis, pterygium keratitis, sjogren's disease, acne rosacea, fryctenosis
syphilis, mycobacterial infection, lipid degeneration, chemical burn, bacterial ulcer, fungal ulcer, herpes simplex infection, shingles infection, protozoan infection, kaposi's sarcoma, Mohren's ulcer, terien marginal degeneration, marginal keratitis Rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoma, scleritis, Stevens-Johnson disease, choroidal radial keratotomy, and corneal graft rejection.

Diseases associated with retinal / choroidal neovascularization include, but are not limited to: diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudofibroelastic xanthomas Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitretis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Yield's disease, Bechets disease, infections that cause retinopathy or choroiditis, putative ocular histoplasmosis, best disease, myopia, congenital structural defects of the optic disc, Stargardt disease, squamitis, chronic retinal detachment, hyperviscosity Consistency syndrome, toxoplasmosis, trauma and complications after laser treatment.

Other diseases include, but are not limited to: rubeosis (
Diseases associated with angulation (angiogenesis), and diseases caused by abnormal growth of vascular or fibrous tissue, including all forms of proliferative vitreoretinopathy.

Chronic inflammation is also associated with pathological angiogenesis. Such disease states, such as ulcerative colitis and Crohn's disease, show histological changes, as well as ingrowth of new blood vessels into inflammatory tissue. Bartonellosis (bacterial infection) found in South America can result in a chronic stage characterized by proliferation of vascular endothelial cells.

Another pathological role associated with angiogenesis is found in atherosclerosis. Plaque formed within the lumen of blood vessels has been shown to have angiogenic stimulatory activity. VEGF expression in human coronary atherosclerotic lesions has been demonstrated by Inoue et al. (1998, specifically incorporated herein by reference). This demonstrates the pathophysiological significance of VEGF in the progression of human coronary atherosclerosis and in the recanalization process in obstructive coronary disease. The present invention provides an effective treatment for such a condition.

One of the most frequent angiogenic diseases of childhood is hemangiomas. In most cases, the tumor is benign and regresses without intervention. In more severe cases, tumors progress to large cavities and invasive tissues and create clinical complications. Hemangionmato, a systemic form of hemangiomas,
ses) have a high mortality rate. There are treatment-resistant hemangiomas that cannot be treated using currently used therapies.

Angiogenesis is also responsible for the damage found in hereditary diseases such as Ausler-Weber-Randue disease, a hereditary hemorrhagic telangiectasia. It is a genetic disease characterized by multiple small hemangiomas, tumors of the blood vessels or lymphatic vessels. Hemangiomas are found in the skin and mucous membranes and often occur by epistaxis (nosebleeds) or gastrointestinal bleeding, and sometimes with pulmonary and hepatic arteriovenous fistulas.

Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also implantation of the blastula after fertilization. Blocking angiogenesis may induce amenorrhea to block ovulation or prevent implantation by blastula.

In wound healing, hyperrepair or fibroplasia can be a detrimental side effect of surgical procedures and can be caused or exacerbated by angiogenesis.
Adhesion is a frequent complication of surgery and leads to problems such as small bowel obstruction.

Diseases and disorders characterized by unwanted vascular permeability can also be treated according to the present invention. These include edema associated with brain tumors, ascites associated with malignancies, Meggs syndrome, pulmonary inflammation, nephrotic syndrome, endocardial effusion and pleural effusion, which are disclosed in WO 98/16551 and incorporated herein by reference. Incorporated.

As described in the following sections, each of the foregoing diseases and disorders, along with all types of tumors, is described, for example, in US Pat. No. 5,712,291 (specifically, (Incorporated by reference), according to the knowledge in the art, the benefits that are effectively treated and integrated by the present invention are the application of an anti-angiogenic strategy to the treatment of angiogenic diseases. Arising from

The antibodies and / or immunoconjugates of the present invention are most preferably utilized in treating tumors. Tumors where angiogenesis is important include malignant and benign tumors, such as neuromas, neurofibromas, trachoma and purulent granulomas. Angiogenesis is particularly prominent in solid tumor formation and metastasis. However, angiogenesis also affects blood-forming tumors (eg, leukemia) and various acute or chronic neoplastic diseases of the bone marrow where unrestricted proliferation of leukocytes occurs (usually anemia, impaired Blood clotting, and with the enlargement of lymph nodes, liver and spleen). Angiogenesis also plays a role in abnormalities in the bone marrow, resulting in leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. In primary tumor angiogenesis, angiogenesis allows cells to enter the blood stream and circulate throughout the body. After the tumor has left the primary site and has colonized the site of the second metastasis, neovascularization must occur before the new tumor can grow and spread. Thus, prevention of angiogenesis may prevent tumor metastasis and may include neoplastic growth at the primary site, allowing for treatment with other therapies, particularly therapeutic-targeting drug constructs ( See below).

The VEGFR2-blocking anti-VEGF antibodies and 2C3-based antibody or immunoconjugate methods provided by the present invention are therefore widely applicable to the treatment of any malignant tumor having a vascular component. When using the antibodies and / or immunoconjugates of the invention for the treatment of tumors, especially vascularized malignancies, the agent may be used alone or
Or for example, chemotherapeutic, radiotherapeutic, apoptotic, anti-angiogenic and / or immunotoxin or coaguligand
Can be used in combination with

[0614] Typical vascularized tumors for treatment are solid tumors, especially cancers, which require vascular components for the supply of oxygen and nutrients. Exemplary solid tumors that can be treated using the present invention include lung, breast, ovary, stomach, pancreas, larynx, esophagus,
Testis, liver, parotid gland, bile duct, colon, rectum, cervix, uterus, endometrium, kidney, bladder,
Examples include, but are not limited to, prostate, thyroid tumors, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma and the like. WO 98/4
5331 is incorporated herein by reference to further illustrate various tumor types that can be effectively treated using anti-VEGF antibodies.

Knowledge of the role of angiogenesis in tumor maintenance and metastasis leads to prognostic indicators for cancers such as breast cancer. The amount of neovascularization found in primary cancers is
Determined by measuring microvessel density in the most intense neovascularization areas in invasive breast cancer. High levels of microvessels were found to correlate with tumor recurrence. Control of angiogenesis by the treatment of the present invention reduces or counteracts such tumor relapses.

The present invention is contemplated for use in treating any patient with a solid tumor. In view of the particular properties of VEGFR2-blocking anti-VEGF antibody-based compositions, the therapeutic agents of the present invention have reduced side effects. Particular advantages result in the maintenance or enhancement of the host immune response to tumors mediated by macrophages and lacking adverse effects on bone tissue. The present invention may therefore be the anti-angiogenic therapy of choice for the treatment of patients with or at risk for developing childhood cancer and osteoporosis and other bone defects.

All malignancies and solid tumors can be treated according to the present invention, but the unconjugated VEGFR2-blocking anti-VEGF and 2V3 antibodies of the present invention are useful in patients with more angiogenic tumors or metastases. Special consideration is given for use in the treatment of patients at risk for

The present invention also provides preventive treatment or prophylaxis.
phylactive) treatment. These aspects of the invention include the ability of the invention to treat patients presenting with a primary tumor that may have metastatic cancer or tumor cells at an early stage of metastatic tumor dissemination. As an anti-angiogenesis strategy, the present invention can also be used to prevent tumor progression in subjects at moderate or high risk for tumor progression, suffering from prognostic testing and / or hereditary cancer Based on relatives.

The conjugated or immunotoxin forms of the VEGFR2-blocking anti-VEGF antibodies and 2V3 antibodies of the invention can destroy or debulk solid tumors (de-bulk).
ing). These aspects of the invention can be used with the unconjugated anti-angiogenic antibodies of the invention, or with other anti-angiogenic approaches.

It will be apparent to those skilled in the art that the immunoconjugate and prodrug forms of the treatment methods of the present invention have the additional advantage of providing a single therapeutic agent with the following two properties: Anti-angiogenic properties and therapeutic properties of the attached drug (eg,
Cytotoxicity, coagulation, apoptosis, etc.). Thus, the conjugated and prodrug-treated forms of the antibodies of the present invention have incredible broad utility across the field of cancer therapy.

The guidance provided herein with respect to patients more appropriate for use in connection with different aspects of the invention suggests that a particular patient profile may assist in selecting a patient for treatment according to the invention. Intended as a teaching. Preselection of a particular patient, or category of patients, in any way negates the utility of the present invention in connection with treating all patients with vascularized tumors, or other angiogenic diseases as described above. do not do. A further consideration is that the attack on the tumor provided by the present invention renders the tumor amenable to further therapeutic treatment, such that subsequent treatment results in an overall synergistic effect or even leads to complete remission or cure.

It is not conceivable that any particular type of tumor would be excluded from treatment using the present invention. However, tumor cell types may be relevant for the use of the present invention in combination with other therapeutic agents, particularly chemotherapeutic agents and anti-tumor cell immunotoxins. The unconjugated and conjugated aspects of the treatment of the present invention include anti-angiogenic effects that inhibit tumor vasculature growth.
The conjugation and prodrug treatment aspects further destroy or close the tumor vasculature. Because the vasculature is substantially or entirely the same in all solid tumors, the methodology of the present invention is broadly applicable to the treatment of all solid tumors, regardless of the particular phenotype or genotype of the tumor cells themselves. It is understood that this is applicable to all or all.

Therapeutically effective doses of VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugate constructs use data from animal models, for example, as shown in the studies detailed herein. And can be easily determined. Laboratory animals bearing solid tumors are often used to optimize the appropriate therapeutic dose before moving to a clinical setting. Such models are known to be very reliable in predicting effective anti-cancer strategies. For example, mice bearing solid tumors (eg, as used in this example) are widely used in preclinical studies. The present inventors have used mouse models accepted in such fields to determine the therapeutic scope of therapeutic agents that provide beneficial anti-tumor effects with minimal toxicity.

When using unconjugated VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies in anti-angiogenic therapy, one of skill in the art will also be able to assist in formulating doses for clinical treatment. Published data can be used. For example, while the antibodies of the invention have different advantages over antibodies in the art, information in the literature regarding treatment with other anti-VEGF antibodies is still relevant for designing or optimizing treatment protocols and doses. , Can be used in combination with the data and teachings of the present application.

For example, Brogstrom et al. (1999), specifically incorporated herein by reference, use MAb A4.6.1 to demonstrate the use of VEGF in breast cancer angiogenesis in vivo. Significance was noted. The 2C3-like antibody of the present invention comprises A4
. While showing comparable or improved anti-tumor responses in comparative studies with 6.1, these antibodies also have significant utility in treating breast cancer. We further recognize that patients with breast cancer are typically women in the middle-aged or elderly population, as will be appreciated by those skilled in the art, where the involvement with osteoporosis is also ,it is obvious. Thus, the VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody of the present invention has the additional advantage of not causing a detrimental effect on bone metabolism and breast cancer patients with osteoporosis or at risk for its progression Is not preferred for use in

The same type of advantage makes VEGFR2-blocking anti-VEGF antibodies and 2C3-based therapeutics the preferred drugs for the treatment of childhood cancer. The need to maintain healthy and substantial bone growth in children with cancer is apparent. VEGFR2 blocking anti-VEGF antibodies (eg, 2C3) do not substantially impair the activity of osteoclasts and cartilage resorbing cells, and since they are important in bone growth, 2C3 may be used in other antibodies (eg, A4.6.1) has an important advantage.

(Blogstrom et al. (1999), specifically incorporated herein by reference) states that MAb A4.6.1 produces significant tumor regression when used in combination with doxorubicin. Reported. This further implies that VEGFR2 blocking anti-V
Supports the combined use of EGF antibodies with conventional cytotoxic or chemotherapeutic agents. Both unconjugated doxorubicin and doxorubicin prodrug formulations are contemplated.

Ferrara and coworkers also reported on the potency and concentration-response of mouse anti-VEGF monoclonal antibodies in tumor-bearing mice, and their implications for human treatment (Mordenti et al., 1999, specifically herein). As a reference). These studies are designed to assess the concentration-response relationship of a mouse anti-VFGF monoclonal antibody so that the effective plasma concentration of a recombinant humanized form of the antibody can be evaluated in cancer patients. Mordenti et al. (19
99) Satisfactory tumor suppression in nude mice is achieved using a dose of mouse antibody, which is effective to maintain the therapeutic antibody for human use in the required effective range. It was concluded that it could easily be applied to human systems to define a clinical dosing regimen. Thus, data from a mouse model that accepts the techniques of the present invention will also include the type of analysis reported in Mordenti et al. (1999), in addition to techniques known to those of skill in the art, as described herein. Can be used to convert to an appropriate human dose.

The results of the preclinical safety assessment of the recombinant humanized form of Genentech's anti-VEGF antibody in monkeys (Ryan et al., 1999, which is specifically incorporated herein by reference) are associated with certain candidate therapeutic agents. Helps to illustrate shortcomings. Although this antibody has pharmacological activity in this animal, in these studies the monkeys were found to undergo superheterotrophic chondrocytes, subchondral bone plate formation, and growth plates.
FIG. 4 shows epiphyseal dysplasia characterized by a dose-related increase in vascular invasion of the rat. Such disadvantages are not evident in the use of VEGFR2-blocking anti-VEGF antibodies and 2C3-based therapeutics. This use does not inhibit VEGF binding and signaling in cartilage resorbing cells and chondrocytes mediated by VEGFR1.

Data from further studies on the preclinical pharmacology, interspecies scaling and tissue distribution of Genentech's humanized monoclonal anti-VEGF antibody are incorporated by Lin et al. (1999, herein incorporated by reference). ). These studies have been performed in mice, rats, monkeys, and rabbits, the latter (rabbits) using ¹²⁵ I-labeled antibodies. Pharmacological data from mice, rats and monkeys were used to estimate the pharmacokinetics of the humanized counterpart using relative allometric comparisons in humans. Thus, appropriate dosage information may be developed for treatment of human pathological conditions such as rheumatoid arthritis, ocular neovascularization and cancer.

A humanized version of the anti-VEGF antibody A4.6.1 was used in clinical trials such as anti-cancer drugs (Brem, 1998; Baca et al., 1997; Presta).
Et al., 1997; each of which is incorporated herein by reference). Therefore, such clinical data may also be considered as a reference source when designing therapeutic doses for VEGFR2-blocking anti-VEGF antibodies and 2C3 treatments of the invention. Although the present invention shows that 2C3 is as effective as A4.6.1 in studies of tumor-bearing mice, the specificity of VEGF for inhibiting only VEGFR2-mediated effects is an advantage. WO 98/45331 is also incorporated herein by reference to illustrate doses of humanized anti-VEGF antibodies that can be used in treatment.

[0632] For the use of conjugated VEGFR2-blocking anti-VEGF antibodies or 2C3-based immunoconjugates in tumor therapy, those skilled in the art will deliver a wide range of therapeutic agents to the tumor vasculature to achieve a beneficial effect. References may be made to the chemical and patent literature on the success. These documents include U.S. Patent Nos. 5,855,866;
Nos. 877,289; 5,965,132; 6,051,230; 6,004,555; 5,776,427; 6,004,554.
No. 6,036,955; and U.S. patent application Ser.
No. 369 (registration fee paid on October 20, 1998)
These are incorporated herein by reference for the purpose of further describing the use of such therapeutic drug-targeting drug constructs.

As is known in the art, there are practical objectives that can be used as guidelines in connection with preclinical trials before proceeding to clinical treatment. But other V to clinical
In view of the prevalence of EGF antibodies, the anti-tumor effects already demonstrated in the accepted models presented herein, and the enhanced safety of current strategies, the present invention Bring the way to therapeutic agents. Thus, preclinical studies can be utilized to select the most advantageous antibodies, doses or combinations.

Any VEGFR2 that produces any consistently detectable anti-angiogenic effect, inhibition of metastasis, tumor vasculature destruction, tumor thrombosis, necrosis and / or general anti-tumor effects
The dose or combined medicament of a blocking anti-VEGF antibody or a 2C3-based antibody or immunoconjugate defines a useful invention. The present invention may also be effective against vascular downstream of tumors. That is, the present invention targets at least a subset of draining blood vessels, and in particular, cytokines released from the tumor
When acting on these blood vessels, they alter their antigenic profile.

[0635] Even in situations where the anti-angiogenic and / or tumor effects of a dose or combination treatment of a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate are toward the lower end of the intended therapeutic range, It is also understood that this treatment may still be equivalent to, or even more effective than, all other known treatments in a particular tumor target or patient setting. It is unfortunately obvious to clinicians that certain tumors and conditions cannot be effectively treated in the medium or long term, but they are at least as effective as other commonly proposed strategies. In some cases, it does not negate the usefulness of the treatment of the present invention.

VEGFR2 blocking anti-VEGF antibody or 2C for treatment of vascularized tumors
In designing an appropriate dose of a 3-based antibody or immunoconjugate construct or combination of therapeutic agents, one of ordinary skill in the art will need to arrive at the appropriate dose for clinical administration.
It can be easily inferred from the common technical knowledge in animal studies and literature described herein. To achieve a dose conversion from animal to human, one of ordinary skill in the art will:
Calculating the mass of drug administered per unit mass of the experimental animal, and preferably,
The difference in body surface area (m ² ) between the experimental animal and the human patient is calculated. All such calculations are well known to those skilled in the art and are routine.

For example, taking a successful dose of 2C3 in a mouse study, and
Applying the standard calculation based on surface area, the effective dose for use in human patients
Is about 1 mg / m^Two~ About 1000mg / m^Two, Preferably about 50 mg / m^Two~about
500mg / m^TwoAnd most preferably about 10 mg / m^Two~ 100mg / m ^Two It is. These doses are based on VEGFR2 blocking anti-VEGF antibody or 2C3-based
Naked antibody and VEGFR2 blocking anti-VEGF antibody or 2C3-based immunoconjugate
Appropriate for coalescence, this dose is suitable for use as an anti-angiogenic agent.
Preferred for use in connection with naked or unconjugated antibodies.

Thus, using this information, we have found that useful low-dose VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugates for human administration are about 1 mg / m ² , 2 mg / M ² , 3 mg / m ² , 4 mg / m ² , 5 mg / m ² , 6
mg / m ² , 7 mg / m ² , 8 mg / m ² , 9 mg / m ² , 10 mg / m ² , 12 m
g / m ² , 15 mg / m ² , 20 mg / m ² , 25 mg / m ² , about 30 mg / m ² ,
35 mg / m ² , 40 mg / m ² , 45 mg / m ² or about 50 mg / m ² ;
And such antibodies or immunoconjugates of useful high doses for human administration is from about ^{600mg / m 2, 650mg / m} 2, 700mg / m 2, 750mg / m 2, 80
^{0mg / m 2, 85mg / m} 2, 900mg / m 2, 925mg / m 2, 950mg
/ M ^2, is 975 mg / m ² or about 1000 mg / m ^2. Useful moderate doses of VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies or immunoconjugates for human administration are about 55 mg / m ² , 60 mg / m ² , 70 mg / m ² , 80 mg / m ² ,
mg / m ² , 90 mg / m ² , 100 mg / m ² , 125 mg / m ² , 150 mg / m ²
m ² , 175 mg / m ² , 200 mg / m ² , 250 mg / m ² , 300 mg / m ²
, 350 mg / m ² , 400 mg / m ² , 450 mg / m ² , 500 mg / m ² , 5
It is contemplated that any dose between ^{25mg / m 2, 550mg / m} 2 or lower doses and high dose, such as about 575 mg / m ^2.

Any particular range or any intermediate value between the stated ranges using any of the exemplary doses recited above is contemplated. VEGFR2 blocking anti-VE
Where a GF antibody or 2C3-based immunoconjugate is used, it is also understood that coagulant immunoconjugates can generally be used at higher doses than toxin immunoconjugates.

Generally, about 10-100 mg / m ² , about 10-90 mg / m ² , about 10-80 m
g / m ^2, about 20 to 100 mg / m ^2, about 20~90mg / m ^2, about 20~80m
g / m ^2, about 30-100 mg / m ^2, about 30~90mg / m ^2, about 30~80m
g / m ² , about 15-100 mg / m ² , about 25-100 mg / m ² , about 35-10
0 mg / m ^2, about 15~90mg / m ^2, about 25~90mg / m ^2, about 35 to 90
A dosage range between mg / m ² or such a dosage range of a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate is preferred. Notwithstanding the scope of these descriptions, it is understood that, in light of the parameters and detailed guidance presented herein, further changes in activity or optimal ranges are within the scope of the invention. .

Thus, in combination with other agents, lower doses may be more appropriate, and in particular, VEGFR2-blocking anti-VEGF antibodies and 2C3 that bind only to VEGFR2
In view of the enhanced safety of base-based antibodies, and even further enhanced safety of VEGFR2-blocking anti-VEGF antibodies and 2C3-based anticoagulants and anti-angiogenic immune conjugates, high doses may still be acceptable That should be understood. The use of human or humanized antibodies (and, optionally, human anticoagulants or anti-angiogenic proteins) further reduces the opportunity for significant toxicity or side effects in healthy tissues, and makes the present invention useful for clinical use. To be more secure.

The intention of the treatment regimes of the invention is to produce a significant anti-tumor effect, while still maintaining doses generally below levels associated with unacceptable toxicity.
In addition to changing the dose itself, dosage regimens can also be employed to optimize the treatment strategy. One treatment protocol is about 1 mg / m ^{2 to} 1000 mg /
m ^2, preferably from about ^{50mg / m 2 ~500mg / m 2} , and most preferably, VEGFR2 blocking anti-VEGF antibody or 2C3-based antibody or immunoconjugate of about 10 mg / m ² ~ about 100 mg / m ^2, or Therapeutic cocktails containing these are administered 1-3 times a week, preferably by intravenous or intramuscular administration, most preferably by intravenous administration.

For administration of a particular dose, one of skill in the art will preferably recognize a pharmaceutically acceptable composition (
(According to FDA standards of sterility, pyrogenicity, purity and general stability) to the patient systemically. Intravenous injection is generally preferred. It is also contemplated to use continuous infusion over a period such as about 1 hour or 2 hours.

[0644] Naturally, prior to extensive use, clinical trials will be performed. The various elements of conducting a clinical trial, including patient treatment and monitoring, are known to those of skill in the art in light of the present disclosure. The following information is provided as general guidelines for use in establishing such a trial.

[0645] Patients selected for a first VEGFR2-blocking anti-VEGF antibody or 2C3-based treatment study will not respond to at least one course of conventional therapy, and physical examination, laboratory techniques, and / or radiography Having an objectively measurable disease as determined by the means. At least two chemotherapy treatments are included in this study
Stop a week ago. If using a mouse monoclonal antibody or antibody portion,
The patient has no history of allergy to mouse immunoglobulins.

A particular advantage is found when using indwelling central venous catheters with triple lumen ports. VEGFR2 blocking anti-V
The EGF antibody or 2C3-based agent is filtered using, for example, a 0.22μ filter and appropriately diluted (eg, with saline) to a final volume of 100 ml. Prior to use, the test sample is also filtered in a similar manner, and its concentration is evaluated before and after filtration by measuring _A280 . Expected recoveries are in the range of 87% to 99%, and then adjustments for protein loss are corrected.

The VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody or conjugate is administered over a period of about 4 to 24 hours, with each patient receiving 2 to 7 days at 2 to 7 day intervals.
Receive ~ 4 injections. Administration may also be performed by constant rate infusion over a period of 7 days. Infusion given at any dose level will depend on any toxicity observed. Thus, if grade II toxicity is reached after any single infusion or for a specific period of time between constant rate infusions, discontinue further doses or stop the constant rate infusion unless the toxicity improves. . Increasing doses of VEGFR2-blocking anti-VEGF antibodies or 2C3-based therapeutics should be used until about 60% of patients show unacceptable grade III or IV toxicity in any category.
Administered to a group of patients. The dose that is 2/3 of this value is defined as the safe dose.

Physical examination, tumor measurements, and laboratory tests are, of course, performed before treatment and at intervals up to one month later. Laboratory tests include whole blood counts, serum creatinine, creatinine kinase, electrolytes, urea, SGOT, nitrogen, bilirubin, albumin, and total serum proteins. Serum samples collected up to 60 days after treatment are evaluated by radioimmunoassay for the presence of antibodies to the administered therapeutic agent and any portion thereof. Immunological analysis of the serum using any standard assay (eg, such as ELISA or RIA)
Enables assessment of pharmacokinetics and clearance of EGFR2-blocking anti-VEGF antibodies or 2C3-based therapeutics.

To assess the anti-tumor response, patients should be tested 48 hours to 1 week after the last infusion and again 30 days after. If there is a palpable disease, the two perpendicular diameters of all the masses
) Should be measured daily during treatment, within one week after treatment is complete, and at 30 days. To measure nonpalpable disease, continuous CT scans can be performed throughout the chest, abdomen, and pelvis at 1 cm intervals, 48 hours to 1 week, and again in 30 days. Tissue samples should also be evaluated histologically and / or by flow cytometry, using biopsies from disease sites or, if appropriate, from blood or body fluid samples.

The clinical response can be defined by acceptable criteria. For example, a complete response may be defined by the absence of any measurable tumor one month after treatment. In contrast, a partial response, one month after treatment, without tumor sites showing growth,
It can be defined by a 50% or more reduction in the sum of the products of the vertical diameters of all evaluable tumor nodules. Similarly, a mixed response may be defined by a 50% or greater reduction in the product of the vertical diameters of all measurable lesions with progression at one or more sites one month after treatment.

In view of the results of the clinical trials described above, even more accurate treatment regimes may be prescribed. Even so, some variation in dosage is
It may be necessary later, depending on the condition of the subject to be treated. The physician responsible for administration will be able to determine the appropriate dose for the individual subject in view of the present disclosure. Such optimizations and adjustments are routinely performed in the art and do not reflect excessive experimental volumes.

G. Combination Therapy Angiogenesis disorders such as arthritis, psoriasis, atherosclerosis, diabetic retinopathy,
When used to treat age-related macular degeneration, Graves' disease, vascular restenosis, hemangiomas and neovascular glaucoma (or other diseases mentioned above) or solid tumors, the present invention provides Can be combined.

The VEGFR2-blocking anti-VEGF antibodies or 2C3-based treatment methods of the present invention
It may be combined with any other commonly used method in the treatment of a particular tumor, disease or disorder indicated by a patient. Certain therapeutic approaches are not known to be harmful per se to the patient's condition, and VEGFR2 blocking anti-VEGF
Unless significantly offsetting the antibody or 2C3-based treatment, its combination with the present invention is contemplated.

In the context of solid tumor treatment, the present invention can be used in combination with classical approaches (eg, surgery, radiation therapy, chemotherapy, etc.). Thus, the present invention provides a combination therapy, wherein the VEGFR2-blocking anti-VEGF antibody or 2C3-based construct is used simultaneously with, before, or after surgery or radiation treatment; A chemotherapeutic, radiotherapeutic, or anti-angiogenic agent, or a targeted immunotoxin or coaguligand,
Before or after that, it is administered to the patient.

The use of the present invention in combination with radiation therapy, radiotherapy drugs, anti-angiogenic drugs, apoptosis-inducing drugs and anti-tubulin drugs is particularly preferred. Many examples of such agents are described above, along with the immunoconjugates of the invention. Any of the agents first described for use as part of a therapeutic conjugate may also be used separately, but may also be used in operable combination with the present invention.

When one or more agents are used in combination with a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapy, the combined results indicate the phase of the effect observed when each treatment is administered separately. There is no need to be additive. While at least an additive effect is generally desired, increased anti-tumor effects beyond one of the single therapies would be beneficial. Also, there is no specific requirement that the combination treatment exhibit a synergistic effect, but this is of course possible and advantageous.

To perform a combined anti-tumor treatment, VEGF was combined with another anti-cancer agent in a manner effective to produce their combined anti-tumor effect in an animal.
Animals are simply administered R2-blocked anti-VEGF antibody or 2C3-based constructs. Thus, the agents are provided in an effective amount and for an effective time to cause their combined presence in the tumor vasculature and their combined action to occur in the tumor environment. To this end, a VEGFR2-blocking anti-VEGF antibody or 2C3-based therapeutic and anti-cancer agent is simultaneously administered to an animal, either in a single composition or in two different compositions using different routes of administration. obtain.

Alternatively, VEGFR2-blocking anti-VEGF antibody or 2C3-based treatment can be given prior to or following anticancer drug treatment, for example, at intervals ranging from minutes to weeks and months. One of skill in the art will recognize that anti-cancer agents and VEGFR2-blocking anti-VEGF antibodies or 2C3-based agents exert advantageous combined effects on tumors.

Most anti-cancer agents can be given prior to VEGFR2-blocking anti-VEGF antibody or 2C3-based anti-angiogenic therapy. However, if a VEGFR2 blocking anti-VEGF antibody or a 2C3-based immunoconjugate is used, the various anti-cancer agents can be administered simultaneously or sequentially.

The general use of a combination of substances in the treatment of cancer is well known. For example,
US Pat. No. 5,710,134, incorporated herein by reference, discloses components that induce necrosis in tumors in combination with non-toxic substances or “prodrugs”. Enzymes released by the necrosis process cleave non-toxic "prodrugs" into toxic "drugs", which lead to tumor cell death. Also, U.S. Pat. No. 5,747,469, which is incorporated herein by reference,
Disclosed is the combined use of p53 and a viral vector encoding a DNA damaging agent. Any such similar approach can be used with the present invention.

In some situations, the duration of treatment is significantly extended (a few days (
2, 3, 4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8)
) Or even several months (elapse of 1,2,3,4,5,6,7 or 8)) may even be desirable. This means that one treatment is intended to substantially destroy the tumor (eg, surgery and chemotherapy) and another treatment is to prevent micrometastases or tumor regrowth ( It is advantageous, for example, in anti-angiogenesis-based treatment situations. Anti-angiogenic agents are carefully administered after surgery,
It should allow for efficient wound healing.

It is also envisioned that more than one administration of a VEGFR2 blocking anti-VEGF antibody or 2C3-based agent or anti-cancer agent will be utilized. These agents can be administered interchangeably every other day or every other week; or a series of VE
A GFR2-blocking anti-VEGF antibody or 2C3-based treatment may be performed, followed by a series of anti-cancer drug therapies. In any case, to achieve tumor regression using combination therapy, all that is required is to deliver both agents in a combined amount effective to exert an anti-tumor effect, regardless of the time of administration. It is to be.

For surgery, any surgical intervention may be performed in combination with the present invention. Any mechanism that induces DNA damage locally in tumor cells in connection with radiation therapy is contemplated, such as, for example, gamma irradiation, X-rays, UV irradiation, microwaves, and even electronic emission. You. Delivery of directed radioisotopes to tumor cells is also contemplated, and is used in the context of targeting antibodies or other targeting means, and preferably, a VEGFR2-blocking anti-VEGF antibody (eg, 2C3). obtain.

[0666] Cytokine treatment has also been demonstrated to be an effective partner in a combined treatment regime. Various cytokines can be used in such a combination approach. Examples of cytokines include: IL-1α
, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7
, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, T
GF-β, GM-CSF, M-CSF, G-CSF, TNFα, TNFβ, LA
F, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, T
MF, PDGF, IFN-α, IFN-β, IFN-γ. The cytokine is administered according to a standard regimen consistent with clinical indicators, such as the condition of the patient, and the relative toxicity of the cytokine. Uteroglobin
n) can also be used to prevent or inhibit metastasis (US Pat. No. 5,696,0
No. 92; incorporated herein by reference).

G1. Chemotherapeutic Agents In certain embodiments, the VEGFR2-blocking anti-VEGF antibodies or 2 of the present invention
C3-based therapeutic agents can be administered in combination with chemotherapeutic agents. A variety of chemotherapeutic agents can be used in the combination treatment methods disclosed herein. Chemotherapeutic agents contemplated by way of example include: adriamycin, dactinomycin, mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol, taxotere, vincristine, vinblastine, vinorerubine, etoposide ( VP-16), 5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa, methotrexate, camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP), aminopterin, combretastatin And their derivatives and prodrugs.

As will be appreciated by those skilled in the art, appropriate doses of chemotherapeutic agents have generally been utilized in clinical treatment, where the chemotherapeutic agents may be used alone or with other chemotherapeutic agents. It is administered in combination with an agent. By way of example only, agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin is
An effective dose that can be widely used to treat cancer and used in clinical applications is 20 mg / m ² every 3 weeks for 5 days for a total of 3 routes. Cisplatin is not absorbed orally and must therefore be delivered via injection, intravenously, subcutaneously, intratumorally, or intraperitoneally.

[0667] Additional useful agents include compounds that interfere with DNA replication, mitosis, and chromosome segregation. Such chemotherapeutic compounds include adriamycin (also known as doxorubicin), etoposide, verapamil, podophyllotoxin and the like. Because of their widespread use in clinical settings for the treatment of neoplasia, these compounds are administered intravenously for adriamycin at 21 day intervals.
In a dose ranging from 5~75mg / m ² to 35~50mg / m ² for etoposide, through bolus injections intravenously or at double the orally intravenous dose,
Is administered.

Agents that disrupt the synthesis and fidelity of polynucleotide precursors may also be used. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU) are preferentially used by neoplastic tissues, making them particularly useful for targeting to neoplastic cells. Although highly toxic 5-FU is applicable in a wide range of carriers, including topical, intravenous administration with doses ranging from 3 to 15 mg / kg / day is generally used.

Exemplary chemotherapeutic agents for combination therapy are listed in Table C. Each agent listed therein is exemplary and not meant to be limiting. One of ordinary skill in the art will be aware of "Remington's Pharmaceutical Science"
es ", 15th edition, Chapter 33 (especially pages 624 to 652). Changes in dosage will probably occur depending on the condition of the subject to be treated. The treating physician will be able to determine the appropriate dose for the individual subject.

[0670]

[Table 4] G2. Anti-Angiogenesis Under normal physiological conditions, humans or animals undergo anti-angiogenesis only in very specific restricted situations. For example, angiogenesis is commonly observed in wound healing, fetal and embryonic development, and formation of the corpus luteum, endometrium and placenta. Uncontrolled (persistent and / or unregulated) angiogenesis is associated with various disease states and occurs during tumor metastasis.

It is believed that both controlled and uncontrolled angiogenesis proceed in a similar manner. Endothelial cells and pericytes (surrounded by basement membrane) form capillaries. Angiogenesis is initiated by erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which along the lumen of the blood vessel, then protrude through the basement membrane. Angiogenic stimulators
Endothelial cells are induced to migrate through the eroded basement membrane. The migrating cells form a "sprout" from the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. Endothelial sprouts combine with each other to form capillary traps that create new blood vessels.

The present invention based on VEGFR2-blocking anti-VEGF antibodies or 2C3 can be used in combination with any one or more anti-angiogenic therapies. Also included are combinations with other factors that inhibit VEGF, including, for example, other neutralizing antibodies (K
im et al., 1992; Presta et al., 1997; Siussat et al., 1993.
Kondo et al., 1993; Asano et al., 1995), soluble receptor constructs (Kendall and Thomas, 1993; Aiello et al., 1995).
Lin et al., 1998; Millauer et al., 1996), tyrosine kinase inhibitors (Siemeister et al., 1998), antisense strategies, RNA aptamers and ribozymes or VEGF receptors for VEGF (Saleh et al., 1996; Cheng et al., 1996; Ke et al., 1998). ; P
arry et al., 1999; each of which is incorporated herein by reference). Variants of VEGF having antagonist properties are also described in WO 98/165.
51, which is specifically incorporated herein by reference.

Antiangiogenic treatment can be based on the supply of anti-angiogenic factors or inhibition of angiogenic factors. Inhibition of angiogenic factors can be achieved by one or more of the methods described for inhibiting VEGF. Here, methods including neutralizing antibodies, soluble receptor constructs, small molecule inhibitors, antisense, RNA aptamers and ribozymes can all be used. For example, antibodies to angiogenesis are disclosed in US Pat. No. 5,520,91.
No. 4 (particularly incorporated herein by reference). Where FGF is associated with angiogenesis, FGF inhibitors may also be used. Some examples include glycosaminoglycans (eg, alkalan sulfate (arc)
Compounds having N-acetylglucosamine which is altered in sequence with 2-O-uronic sulphonic acid as their repeating unit, including H. sulphate. Such compounds are described in U.S. Patent No. 6,028,061, specifically incorporated herein by reference, and can be used herein.

A number of tyrosine kinase inhibitors are currently known that are useful in treating angiogenesis, as is prominent in various disease states. These include, for example, 4-aminopyrrole [2,3-d] pyrimidine of U.S. Patent No. 5,639,757, which is specifically incorporated herein by reference. This is also
It can be used in combination with the present invention. Further examples of organic molecules that can mediate tyrosine kinase signaling via the VEGDR2 receptor are quinazoline compounds and US Pat. No. 5,792,771 (specifically, For the purpose of describing further combinations).

Other chemical classes of compounds have also been shown to inhibit angiogenesis,
And it can be used in combination with the present invention. For example, US Pat. No. 5,972,9
No. 22 (specifically, angiogenic 4,9 (11) -steroids and C21-oxidized steroids) described in No. 22 (incorporated herein by reference) can be used in combination therapy. Can be used. US Patent 5,712,291
No. 5,593,990, each of which is incorporated herein by reference, describes thalidomide and related compounds, precursors, analogs, metabolites and hydrolysates. These can also be used in combination with the present invention to inhibit angiogenesis. U.S. Patent Nos. 5,712,291 and 5,5
The compound in 93,990 can be administered orally. Additional exemplary anti-angiogenic agents that are useful for combination therapy are listed in Table D. Each agent listed here is an example and is not meant to be limiting.

[0676]

[Table 5] Certain preferred compounds for use in inhibiting angiogenesis are angiostatin, endostain, vasculostatin (v
asculostatin, canstatin and maspin. Such agents are described above in combination with the immunoconjugate of the invention. However, they can be used in combination, but form conjugates. Another preferred agent described above in conjugate form is also angiopoietin (specifically, angiopoietin-2), which is intended for use in combination with the present invention.

Certain anti-angiogenic treatments have previously been shown to cause tumor regression,
Bacterial polysaccharide CM101 and antibody LM609 are included. CM101 is a bacterial polysaccharide, which is well characterized in its ability to induce neovascular inflammation in tumors. CM101 binds and cross-links receptors expressed on dedifferentiated endothelium that stimulate activation of the complement system. It also
Initiate a cytokine-driven inflammatory response that selectively targets tumors. It is a unique anti-pathological angiogenic agent that down-regulates expressed VEGF and its receptor. CM101 is currently in clinical trials as an anticancer agent and can be used in combination therewith.

[0678] Thrombospondin (TSP-1) and platelet factor 4 (PF4) can also be used in conjunction with the present invention. Both are angiogenesis inhibitors that associate with heparin and also platelet α-
Found in granules. TSP-1 is a component of the extracellular matrix,
It is a large 450 kDa multidomain glycoprotein. TSP-1 is HS
It binds to many proteoglycan molecules found in the extracellular matrix, including PG, fibronectin, laminin, and various types of collagen. TS
P-1 inhibits endothelial cell migration and proliferation in vitro and angiogenesis in vivo. TSP-1 can also suppress the malignant phenotype and tumorigenesis of transformed endothelial cells. The tumor suppressor gene p53 has been shown to directly regulate TSP-1 expression, so that loss of p53 activity is
It results in a dramatic decrease in SP-1 production and a concomitant increase in tumor-initiated angiogenesis.

PF4 is a member of the CXC ELR family of chemokines, 70aa
There are proteins, which can potently inhibit endothelial cell proliferation in vitro and angiogenesis in vivo. PF4 administered intratumorally or delivered by an adenoviral vector can result in inhibition of tumor growth.

[0680] Interferons and metalloproteinase inhibitors are two other classes of naturally-occurring angiogenesis inhibitors that can be used in conjunction with the present invention. Although the anti-endothelial activity of interferon has been known since the early 1980's, the mechanism of inhibition is not yet clear. Their ability to inhibit endothelial cell migration and their anti-angiogenic activity in vivo, which are fearfully mediated by their ability to inhibit the production of angiogenic promoters by tumor cells, It is known to have. Vascular tumors are specifically sensitive to interferons, for example, growing hemangiomas can be successfully treated with IFNa.

Tissue inhibitors of metalloproteinases (TIMPs) are a family of inhibitors of naturally occurring matrix metalloproteases (MMPs)
This can also inhibit angiogenesis and can be used in combination treatment protocols. MMPs play an important role in the angiogenesis process. Because they degrade the matrix through which endothelial cells and fibroblasts migrate when expanding or remodeling the vascular network. Indeed, one member of MMP, MMP-2, has been shown to associate with activated endothelium, possibly via integrin αvβ3 for this purpose. If this interaction is disrupted by fragments of MMP-2, angiogenesis is then down-regulated and inhibited in tumor growth.

There are many pharmacological agents that inhibit angiogenesis, any one or more of which can be used in combination with the present invention. These materials include AGM-1470 / T
NP-470, thalidomide, and carboxamidotriazole (CAI)
Is mentioned. Fumagillin was found to be a potent inhibitor of angiogenesis in 1990, and since then synthetic analogs of fumagillin, AGM-1470 and TNP-470, have been developed. Both of these drugs inhibit endothelial cell proliferation in vitro and angiogenesis in vivo. T
NP-470 has been studied extensively in human clinical trials, and its data suggest that long-term administration is optimal.

Thalidomide, which was originally used as a sedative, was found to be a potent teratogen and was discontinued. In 1994, thalidomide was found to be an angiogenesis inhibitor. Thalidomide is currently being clinically tested as an anticancer drug and treatment of vascular eye diseases.

[0684] CAI is a low molecular weight synthetic inhibitor of angiogenesis, which acts as a calcium channel blocker, preventing actin reorganization, endothelial cell migration and spreading on collagen IV. CAI inhibits neovascularization at physiologically achievable concentrations and is well tolerated orally by cancer patients. CAI
A clinical trial with has resulted in disease stabilization in 49% of cancer patients with progressive disease prior to treatment.

[0686] Cortisone in the presence of heparin or heparin fragments has been shown to inhibit tumor growth in mice by blocking endothelial cell proliferation. The mechanism involved in the additive inhibitory effects of steroids and heparin is not clear, but it is believed that heparin can increase steroid uptake by endothelial cells. This mixture has been shown to increase the lysis of the basement membrane beneath newly formed capillaries, and this is also the result of additive angiostasis.
tic) is a possible explanation for the effect. Heparin-cortisol conjugates also have potent angiostatic and antitumor activity in vivo.

[0686] Further specific angiogenesis inhibitors (anti-invasive factors (Anti-Invas
iv Factor), retinoic acid and paclitaxel (U.S. Pat.
No. 716,981; incorporated herein by reference); AGM-1470 (
Ingber et al., 1990; incorporated herein by reference); Shark cartilage extract (US Pat. No. 5,618,925; incorporated herein by reference).
Anionic polyamide oligomers or anionic polyurea oligomers (US Pat. No. 5,593,664; incorporated herein by reference); oxindole derivatives (US Pat. No. 5,576,330). Estradiol derivatives (US Pat. No. 5,5; incorporated herein by reference));
And thiazole pyrimidine derivatives (US Pat. No. 5,599,813; incorporated herein by reference), but not limited thereto. No.) are also contemplated for use as anti-angiogenic compositions for use in combination with the present invention.

Compositions comprising an antagonist of α _v β ₃ integrin may also be used in combination with the present invention to inhibit angiogenesis. U.S. Pat. No. 5,766,591 (
RGD-containing polypeptides and their salts, including cyclic polypeptides, are suitable examples of α _v β ₃ integrin antagonists, as disclosed in U.S. Pat.

The antibody LM609 against α _v β ₃ integrin also induces tumor regression. Integrin α _v β ₃ antagonists (eg, LM609) induce apoptosis of angiogenic endothelial cells, leaving quiescent blood vessels unaffected.
LM609 or other α _v β ₃ antagonists also use α _v β ₃ and MMP-2 (MM
P-2 is a proteolytic enzyme that is thought to play an important role in the migration of endothelial cells and fibroblasts). US Pat. No. 5,753,230 is hereby incorporated by reference to describe antibodies to α _v β ₃ (vitronectin α _v β ₃ ) for combining with the present invention to inhibit angiogenesis. Is specifically referred to in

[0689] Apoptosis of the angiogenic endothelium in this case may have a cascading effect on the rest of the vascular network. Inhibiting the complete response of the tumor vascular network to signals that attempt to spread the tumor actually initiates partial or complete disruption of the vascular network, leading to tumor cell death and tumor This results in a reduction in volume. Endostatin and angiostatin can function in a similar manner. LM609
Does not affect quiescent blood vessels but can cause tumor regression,
It strongly suggests that not all blood vessels in the tumor need to be targeted for treatment in order to obtain an anti-tumor effect.

[0690] Other methods of therapeutic treatment based on altering signaling through the Tie2 receptor can also be used with the present invention, for example, using a soluble Tie2 receptor that can block Tie2 activation (Lin et al., 1998). It can be used in combination. Delivery of such constructs using recombinant adenovirus gene therapy has been shown to be effective in treating cancer and reducing metastasis (Lin et al.,
1998).

G3. Apoptosis Inducing Factors VEGFR2-blocking anti-VEGF antibodies or 2C3-based therapeutics can also be advantageously combined with methods for inducing apoptosis. Various apoptosis-inducing factors are described above in combination with the immunoconjugates of the invention. Any such apoptosis-inducing factor can be used in combination with the present invention without being linked to the antibody of the present invention.

In addition to the apoptosis-inducing factors described above as immunoconjugates, a number of oncogenes that inhibit apoptosis, ie, programmed cell death, have been described. Exemplary oncogenes in this category include bcr-abl, bcl-2 (bc
1-1, different from cyclin D1; GenBank accession number M14745, X0
6487; U.S. Patent Nos. 5,650,491; and 5,539,094; each of which is incorporated herein by reference) and Bcl-xl, Mcl-.
Family members including, but not limited to, 1, Bak, A1, A20. Overexpression of bcl-2 was first discovered in T-cell lymphoma. bcl-2 functions as an oncogene by binding to Bax, a protein in the apoptotic pathway, and inactivating Bax. Inhibition of bcl-2 function prevents Bax inactivation and allows the apoptotic pathway to proceed.

Inhibition of this class of oncogenes (eg, using antisense nucleotide sequences) is contemplated for use in the present invention in situations where enhanced apoptosis is desired (US Pat. No. 5,650, 491; 5,539,094 and 5,583,034; each of which is incorporated herein by reference).

G4. Immunotoxins and Coaguligands The treatment methods based on anti-aminophospholipid conjugates of the invention can be used in combination with other immunotoxins and / or coaguligands, where the targeting moiety ( For example, antibodies or ligands) are directed to relatively specific markers of tumor cells, tumor vasculature or tumor stroma. In common with the chemotherapeutic and anti-angiogenic agents discussed above, the combined use of other targeted toxins or coagulants is generally additive, and more pronounced than additive. Large, or even synergistic, results in antitumor.

Generally speaking, the antibodies or ligands for use in these further aspects of the invention preferably recognize accessible tumor antigens, which preferentially
Or, specifically, it is expressed at the tumor site. The antibody or ligand also preferably exhibits high affinity properties; and the antibody, ligand, or conjugate thereof,
Normal cells that sustain survival (eg, heart, kidney, brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenal gland, muscle, nerve fibers, pancreas, skin, or other survival in the human body) (Organs or tissues to be maintained) without significant in vivo side effects on one or more tissues selected from the group. As used herein,
The term “significant side effects” refers to an antibody, ligand or antibody binding that, when administered in vivo, produces only negligible or clinically manageable side effects (eg, side effects commonly encountered during chemotherapy). Refers to the body.

[0696] At least one binding region of these second anti-cancer agents used in combination with the present invention can deliver toxins or clotting factors to the tumor region (ie, can be localized within the tumor site). It is. Such targeting agents may be directed against components of the tumor cells, tumor vasculature or tumor stroma. The targeting agent generally binds to a surface-expressed, surface-accessible, or surface-localized component of the tumor cell, tumor vasculature, or tumor stroma. However, once the destruction of the tumor vasculature and tumor cells begins, the internal components are released, allowing for further targeting of virtually any tumor component.

[0697] A number of tumor cell antigens have been described, any of which can be used as a target in connection with the combined aspects of the invention. A suitable tumor cell antigen for targeting additional immunotoxins and coag ligands is antibody B3 (US Pat.
ATCC HB 1, 242,813; incorporated herein by reference;
0573); KSI / 4 (U.S. Pat. No. 4,975,369; incorporated herein by reference; vectors NRRL B-18356 and / or NRRL).
B-18357); 260F9 (ATCC HB8
488); and D612 (US Pat. No. 5,183,756; incorporated herein by reference; ATCC HB 9796). One of skill in the art will be able to identify other suitable cell lines that produce anti-tumor cell antibodies,
Any later ATCC catalog may also be consulted.

For tumor vasculature targeting, the targeting antibody or ligand is often
Whether expressed by or adsorbed to intratumoral blood vessels of an angiogenic tumor;
Binds to a marker induced thereon or otherwise located there. Appropriately expressed target molecules include, for example, endoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA (Liu et al., 19
97), TIE, LAM-1 reactive ligand, VEGF / VPF receptor, FGF receptor, α _v β ₃ integrin, pleiotropin (pleiotr)
opin), and endosialin. Suitable targets to be adsorbed include, for example, VEGF, FGF, TGFβ, HGF, PF4
, PDGF, TIMP, ligands that bind TIE, and targets such as tumor-associated fibronectin isoforms. For example, antigens that can be naturally and artificially induced by cytokines and coagulants can also be targeted; ligands responsive to ELAM-1, VCAM-1, ICAM-1, LAM-1, such as: Endoglin and also MHC class II (eg, IL-1, TNF-
cytokine-inducible by α, IFN-γ, IL-4 and / or TNF-β); and E-selectin, P-selectin, PDGF and ICAM-
1 (eg, coagulant-induced by thrombin, factor IX / IXa, factor X / Xa and / or plasmin).

The following patents and patent applications relate to the preparation and use of immunotoxins directed against expressed, adsorbed, induced, or localized markers of tumor vasculature. For the purpose of further complementing the teachings, it is specifically incorporated herein by reference: US Application Ser. No. 08 / 482,369 (199).
U.S. Patent Nos. 5,855,866 and 5,
Nos. 965,132, 6,051,230, 6,004,555, 5,877,289, 6,004,554, and 5,776,427.
Nos. 5,863,538, 5,660,827 and 5,03
6,955.

Additional tumor vascular targeting compositions and methods include the use of these target aminophospholipids, such as phosphatidylserine and phosphatidylethanolamine, which have recently been found to be accessible and specific markers of tumor blood vessels. Including. Administration of the anti-aminophospholipid antibody alone is sufficient to induce thrombosis and tumor regression. Thus, the invention can use non-conjugated, anti-phosphatidylserine and / or phosphatidylethanolamine antibodies; or immunoconjugates of such antibodies.

The following provisional application is specifically incorporated herein by reference for the purpose of still further complementing the teachings of the present invention regarding the preparation and use of anti-aminophospholipid antibodies and immunotoxins: Application Nos. 60 / 092,672 (filed on July 13, 1998) and 60 / 092,589 (filed on July 13, 1998)
. Application No. 60 / 092,589 further discloses aminophospholipid binding protein conjugates (e.g., for use in delivering toxicants and coagulants to tumor blood vessels, and for increasing thrombosis and tumor regression). For the purpose of still further complementing the teachings of the present invention relating to the use of annexin conjugates), it is specifically incorporated herein by reference.

Suitable tumor stroma targets include components of the tumor extracellular matrix or stroma,
Or components bound therein, including: basement membrane markers, IV
Type collagen, laminin, heparan sulfate, proteoglycans, fibronectin, activated platelets, LIBS and tenascin. A preferred target for such use is RIBS.

The following patents and patent applications are specifically incorporated by reference herein for the purpose of still further complementing the teachings of the present invention regarding the preparation and use of agents that target tumor stroma. No. 5,877,289 and U.S. patent application Ser. No. 08 / 482,369 (U.S. Pat. , Issue; registration fee paid);
Nos. 08 / 485,482; 08 / 487,427 (U.S. Pat.
No. 08 / 479,733 (U.S. Pat. No. 5,877,28).
No. 9/08 / 472,631 and 08 / 479,727 and 08 / 481,904 (US Pat. No. 6,036,955).

The second anti-cancer therapeutic agent is any of the cytotoxic or otherwise anti-cellular agents described herein for use in VEGFR2-blocking agents, anti-VEGF antibodies or 2C3-based immunotoxins. An agent can be operably coupled to the agent. However, suitable anticellular agents also include radioisotopes. Preferred are toxin moieties such as ricin A chain and deglycosylated A chain (dgA) or even gelonin. Any one or more angiopoietins, or fusions thereof, may also be used as a second immunoconjugate for combination therapy.
Can be used as part of

The second targeted agent for any use in the present invention can include a targeted component (ie, a coag ligand) that can promote coagulation. Here, the targeting antibody or ligand can be any factor that directly or indirectly (eg, via another antibody) stimulates coagulation, directly or indirectly (VEGFR2-blocking agent, anti-VEGF (Including any of the factors described herein for use in antibodies or 2C3-based coag ligands). Preferred coagulation factors for such use are tissue factor (TF) and TF derivatives (eg, truncated TF (tTF), dimeric and multimeric TFs, and mutant TFs deficient in factor VII activating ability. ).

An effective dose of an immunotoxin and coaguligand for combined use in the treatment of cancer, when administered via the IV route at a frequency of about once per week.
It is between 1 mg / kg and about 2 mg / kg, and preferably between about 0.8 mg / kg and about 1.2 mg / kg. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The physician responsible for administration will determine the appropriate dose for the individual subject.

G5. ADEPT and Prodrug Treatment The VEGFR2 blocking agent, anti-VEGF antibody or 2C3-based antibody of the invention can be used in combination with a prodrug, wherein the VEGFR2 blocking agent, anti-VEGF antibody or 2C3-based Are operably associated with a prodrug activating component (eg, a prodrug activating enzyme that converts a prodrug to a more active form only upon contact with the antibody). This technology is generally referred to as "AD
EPT "and are described, for example, in WO 95/13095; WO 97/2691.
8, WO 97/24143 and U.S. Pat. Nos. 4,975,278 and 5,
No., 568,568, each of which is specifically incorporated herein by reference.

As used herein, the term “prodrug” refers to reduced cytotoxicity or other cytotoxicity to target cells (including tumor vascular endothelial cells) as compared to the parent drug on which the prodrug is based. Refers to a precursor or derivative form of a biologically or pharmaceutically active substance that exerts an anticellular effect. Preferably, the prodrug or precursor form exerts a significant reduction, or more preferably, exerts a negligible cytotoxic or anti-cellular effect as compared to the "native" or parent form. A "prodrug" may be activated or converted to give a more active parent form of the drug.

The technical abilities to make and use a drug are within the skill of the art. Will
(1986) and Stella et al. (1985) are each specifically incorporated herein by reference for the purpose of further providing descriptions and techniques for methods of making and using various prodrugs. Exemplary prodrug constructs that may be used in the context of the present invention include, but are not limited to, phosphate-containing prodrugs (US Pat. No. 4,975,278),
Thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-based prodrugs (US Pat. Nos. 5,660,829; 5,587,1)
No. 5,405,990; WO97 / 07118), D-amino acid modified prodrugs, glycosylated prodrugs (U.S. Pat. Nos. 5,561,119; 5,646,298; No. 4,904,768, No. 5,041,4
No. 24), β-lactam containing prodrugs, optionally substituted phenoxyacetamide containing prodrugs (US Pat. No. 4,975,278), optionally substituted phenylacetamide containing prodrugs and even 5 -Fluorocytosine (U.S. Patent No. 4,975,278) and 5-fluorouridine prodrugs and the like, each of which is specifically incorporated herein by reference.

The types of therapeutic or cytotoxic drugs that can be used in the form of a prodrug are substantially limited. More cytotoxic agents are preferred for such forms of delivery,
For example, it is preferred over the delivery of coagulants (less preferred for use as prodrugs). Designing the construct so that the prodrug is substantially inactive and the "released" or activated drug has activity for a substantial or intended purpose All that is required in the formation of a prodrug.

Various improvements on the original prodrug are also described in WO 95/03380;
EP 751,144 (anthracycline); WO 97/07097 (cyclopropylindole); and as disclosed in WO 96/16969
Known and intended for use with the specification. For example, prodrugs with reduced Km are described in US Pat. No. 5,621,002, which is specifically incorporated herein by reference, which may be used in the context of the present invention. Prodrug therapy performed intracellularly is also known, as exemplified in WO 96/03151, which is specifically incorporated herein by reference, and may be practiced using the present specification. .

For use in ADEPT, an agent that activates or converts the prodrug to a more active drug is operably linked to a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody. Thus, this VEGFR2-blocking anti-VEGF antibody or 2C3-like antibody localizes the prodrug conversion capacity within the site of angiogenesis, preferably within the tumor vasculature and stroma, and the active drug is produced only in such regions. And is not produced in the circulation or in healthy tissue.

Enzymes that can bind to a VEGFR2-blocking anti-VEGF antibody or 2C3-like antibody and function in prodrug activation include alkaline phosphatase for use in combination with phosphate-containing prodrugs (US Pat. No. 4,975,975). 278)
Arylsulfatases for use in combination with sulfate-containing prodrugs (US Pat. No. 5,270,196); peptidases and proteases (eg, Serratia protease, thermolysin, subtilisin) for use in combination with peptide-based prodrugs , Carboxypeptidase (U.S. Patent Nos. 5,660,829; 5,587,161; 5,405,99).
No. 0) and cathepsins (including cathepsins B and L)); D-alanyl carboxypeptidase for use in combination with D-amino acid modified prodrugs; carbohydrate-cleaving enzymes for use in combination with glycosylated prodrugs ( For example, β-galactosidase and neuraminidase) (U.S. Pat. No. 5,561)
No. 5,646,298); β-lactamases for use in combination with β-lactam containing prodrugs; in combination with drugs derivatized at the amino nitrogen with a phenoxyacetamide or phenylacetamide group. (Eg, penicillin V amidase (US Pat. No. 975,278) or penicillin G amidase) for use as well as prodrugs based on 5-fluorocytosine (US Pat. No. 4,975,278). Cytosine deaminase for use in combination (U.S. Pat. Nos. 5,338,678; 5,545,548) include, but are not limited to, each of which is herein incorporated by reference. It is particularly incorporated by reference.

[0714] Antibodies with enzymatic activity (also known as catalytic antibodies or "abzymes") are also
It can be used to convert a prodrug into an active drug. Therefore, VEGF
Abzymes based on R2 blocking anti-VEGF antibodies or 2C3-like antibodies form another aspect of the invention. The technical ability to produce abzymes has also been described by Massey et al.
1987), which is specifically incorporated herein by reference for the purpose of supplementing the teachings of this abzyme, is within the skill of the art. Catalytic antibodies capable of catalyzing prodrug degradation at carbamate positions (eg, nitrogen mustard aryl carbamates) are further contemplated, as described in EP 745,673, which is specifically incorporated herein by reference. You.

H. Diagnostics and Imaging The present invention further provides in vitro and in vivo diagnostics and imaging. Such methods may provide diagnostic, prognostic or image information for any angiogenic disease (illustrated by arthritis, psoriasis and solid tumors, but including all angiogenic diseases disclosed herein). Is applicable for use in generating. Outside of the field of tumor diagnostics and imaging, these aspects of the invention relate to in vitro diagnostic tests (preferably, where the sample is obtained non-invasively and can be tested in a high-throughput assay, and / or It is most preferred for use in ambiguity and confirmation in whichever clinical diagnosis is desired).

H1. Immunodetection Methods and Kits In yet a further embodiment, the present invention provides a method for binding, purifying, removing, otherwise generally detecting VEGF, and for diagnosing an angiogenic disease. ,
It relates to an immunodetection method. VEGFR2-blocking anti-VEGF antibodies of the invention (eg, 2
C3) is VEGF in vivo (described below) in isolated tissue samples, biopsies or swabs, and / or in homogenized tissue samples.
Can be used to detect Such immunodetection methods have obvious diagnostic utility, but also have application to non-clinical samples (eg, in titration of antigen samples, etc.).

The steps of various useful immunodetection methods are described in the scientific literature (eg, Nakamura et al.,
(1987, incorporated herein by reference)). In general, the immunobinding method involves obtaining a sample suspected of containing VEGF, and subjecting the sample to a VEGFR2-blocking anti-VEGF antibody (eg, 2C3) under conditions effective to permit the formation of an immune complex. And contacting with. In such a method, the antibody can be bound to a solid support, for example, in the form of a column matrix, and a sample suspected of containing VEGF is applied to the immobilized antibody.

More preferably, the immunobinding method comprises a method for detecting or quantifying the amount of VEGF in a sample, the method comprising detecting or detecting any immune complexes formed during the binding process. Requires quantification. Here, a sample suspected of containing VEGF is obtained, and the sample is contacted with an antibody according to the specification, and then the amount of immune complex formed under certain conditions is detected or quantified.

The biological sample to be analyzed can be any sample suspected of containing VEGF, and is generally from an animal or patient suspected of having an angiogenic disease. The sample can be a tissue section or specimen, a biopsy, swab or smear test sample, a homogenized tissue extract, or an isolated or purified form of such.

Contacting a selected biological sample with the antibody under conditions effective to allow for the formation of an immune complex (primary immune complex) and for a time sufficient to allow it. Generally simply adds the antibody composition to the sample and all VEGF present and the antibody form an immune complex (ie, bind)
Is to incubate the mixture for a sufficiently long time. After this point,
The sample-antibody composition (eg, tissue section, ELISA plate, dot blot, or Western blot) is generally washed to remove any non-specifically bound antibody species and to remove Only specifically bound antibodies can be detected.

The detection of immune complexes is well known in the art and can be achieved through the application of a number of approaches. These methods are generally based on the detection of a label or marker (eg, any radioactive, fluorescent, biological or enzymatic tag or label known in the art). U.S. Pat. Nos. 3,817,837; 3,850,752; and 3,939,3 U.S. Pat.
No. 50; 3,996,345; No. 4,277,437; No. 4,275.
No. 149 and 4,366,241, each of which is incorporated herein by reference. The use of an enzyme that produces a colored product upon contact with a chromogenic substrate is generally preferred. Secondary binding ligands, such as a second antibody or a biotin / avidin ligand binding arrangement, can also be used, as is known in the art.

VEGFR2-Blocking Anti-VEGF Antibodies Used in Detection (eg, 2C3)
As such, it can be linked to a detectable label, which is then easily detected, allowing the amount of primary immune complex in the composition to be determined.

Preferably, this primary immune complex is detected by a second binding ligand having a binding affinity for an antibody of the invention. In this case, the second binding ligand can be linked to a detectable label. This second binding ligand itself is
It is often an antibody, and may therefore be referred to as a "secondary" antibody. The primary immune complex is contacted with the labeled secondary binding ligand or antibody under conditions effective and for a time sufficient to allow for the formation of a secondary immune complex. You.
This secondary immune complex is then generally washed to remove any non-specifically bound labeled secondary antibody or ligand, and then the remaining label in the secondary immune complex is removed. Is detected.

A further method involves the detection of a primary immune complex by a two-step approach. First
A second binding ligand (e.g., an antibody) that has a binding affinity for the antibody is used to form a secondary immune complex as described above. After washing, the secondary immune complex comprises a third binding ligand or antibody having a binding affinity for the second antibody;
The contact is made under conditions effective to allow the formation of an immune complex (tertiary immune complex) and for a time sufficient to allow it. This third ligand or antibody is linked to a detectable label that allows for detection of the tertiary immune complex so formed. This system can provide signal amplification if desired.

In clinical diagnosis or monitoring of patients with angiogenic diseases, VEGF
Detection of, or an increase in the level of VEGF compared to the level in a corresponding biological sample from a normal subject is indicative of a patient with an angiogenic disease.

However, as is known in the art, such clinical diagnosis will not be performed in isolation on the basis of this method. One of skill in the art is very familiar with distinguishing between significant expression of a biomarker (indicating a positive identification) and low or background expression of the biomarker. In fact, background expression levels are often used to form a "cutoff", and an increase in staining above this cutoff is scored as significant or positive.

H2. Imaging These aspects of the invention are preferred for use in tumor imaging methods and combined tumor treatment and imaging methods. VEGFR2-blocking anti-VEGF antibodies or antibodies based on 2C3 conjugated to one or more detectable agents may be used in imaging of tumors themselves or in pre-imaging of tumors to provide reliable prior to treatment. It is envisaged to form an image with a characteristic. Such compositions and methods also include any other angiogenic disease or condition, particularly non-malignant tumors, atherosclerosis,
And internal imaging can be applied to the imaging and diagnosis of conditions that are desired for diagnostic or prognostic purposes or for designing treatments.

A VEGFR2-blocking anti-VEGF antibody or 2C3-based imaging antibody generally includes a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody operably attached or conjugated to a detectable label. "Detectable labels" are compounds or elements that can be detected due to their specific functional properties, or chemical characteristics, the use of which makes the components to which they are attached detectable, and If so, it can be further quantified. There is a need for labels that can be detected using non-invasive methods in antibody conjugates for in vivo diagnostic protocols or “imaging methods”.

Many suitable imaging agents are known in the art, such as methods of attaching them to antibodies and binding ligands (eg, US Pat. No. 5,021,236)
No. 4,472,509. Both are incorporated herein by reference). Certain attachment methods include, for example, the use of metal chelate conjugates using an organic chelator such as DTPA attached to the antibody (US Pat.
, 472, 509). Monoclonal antibodies can also be reacted with the enzyme in the presence of a binder such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these binders or by reaction with isothiocyanates.

An example of a detectable label is a paramagnetic ion. In this case, suitable ions include: chromium (III), manganese (II), iron (III),
Iron (II), cobalt (II), nickel (II), copper (II), neodymium (I
II), samarium (III), ytterbium (III), gadolinium (I
II), vanadium (II), terbium (III), dysprosium (III
), Holmium (III), and erbium (III), with gadolinium being particularly preferred.

[0731] Ions useful in other situations (eg, x-ray imaging) include, but are not limited to, lanthanum (III), gold (III), lead (II), and especially bismuth (III). Fluorescent labels include rhodamine, fluorescein, and renographin. Rhodamine and fluorescein are often linked via an isothiocyanate intermediate.

For radioisotopes for diagnostic applications, suitable examples include the following:
RU:¹⁴carbon,⁵¹chromium,³⁶chlorine,⁵⁷cobalt,⁵⁸Cobalt, copper⁶⁷,¹⁵²Eu, moth
Lium⁶⁷,^ThreeHydrogen, iodine^{one two Three},Iodine¹²⁵,Iodine¹³¹,indium¹¹¹,⁵⁹iron,^{Three Two} Phosphorus, rhenium¹⁸⁶,rhenium¹⁸⁸,⁷⁵Selenium,³⁵Sulfur, technetium^99m,
And yttrium⁹⁰.¹²⁵I is often used in certain embodiments
Preferred for, and technetium^99mAnd indium¹¹¹Also often
Preferred because they are low energy and suitable for detection over a wide range
.

[0733] Radiolabeled VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies for use in the present invention can be generated by methods well known in the art. For example, intermediate functional groups often used to attach radioisotope metal ions to antibodies are diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA).

The monoclonal antibodies can also be iodinated by contacting with a chemical oxidant such as sodium or potassium iodide and sodium hypochlorite, or an enzyme oxidant such as lactoperoxidase. Antibodies according to the present invention may be prepared by reducing pertechnate with a solution of divalent tin, for example, by a ligand exchange process, chelating the reduced technetium on a Sephadex column, and applying the antibody to the column. by, can be labeled with ^{technetium-99m;} Kamata by direct labeling techniques, e.g. pertechnetate, a reducing agent such as SNCl _2, sodium - incubating a buffer solution such as potassium phthalate solution, and the antibody By
It can be labeled.

Any of the above described types of detectably labeled VEGFR2-blocking anti-VEGF antibodies or 2C3-based antibodies can be used in the imaging or combined imaging and treatment aspects of the invention. These are equally suitable for use in in vitro diagnostics. Dosages for in vivo imaging embodiments will generally be less than for therapeutic use, but will also depend on the age and weight of the patient. One dose should be sufficient.

In vivo diagnostic or imaging methods generally involve administering to a patient a diagnostically effective amount of a VEGFR2-blocking anti-VEGF antibody or 2C3-based antibody conjugated to a marker that is detectable by a non-invasive method. Process. The antibody-marker conjugate localizes to VEGF in the tumor and is left for a time sufficient to bind.
The patient is then exposed to a detection device to identify a detectable marker, thereby forming an image of the tumor.

H3. Diagnostic Kits In yet a further embodiment, the present invention provides a diagnostic kit for use with the immunodetection and imaging methods described above, including both an immunodetection kit and an imaging kit. provide. Thus, VEGFR2 blocking anti-VEGF antibodies (eg, 2C3)
Is provided in a kit, typically in a suitable container.

For immunodetection, the antibody may be bound to a solid support (eg, a well of a microtiter plate), but an antibody solution or a powder for reconstitution is preferred. The immunodetection kit preferably includes at least a first immunodetection reagent. The immunodetection reagents of the kit can take any one of a variety of forms, including a detectable label associated with or bound to a given antibody. A detectable label associated with or associated with the secondary binding ligand is also contemplated. An exemplary secondary ligand is a secondary antibody that has a binding affinity for the first antibody.

[0739] Further suitable immunodetection reagents for use in the present kit are a secondary antibody having a binding affinity for a first antibody, a third antibody having a binding affinity for the second antibody. The third antibody comprises a two-component reagent, both of which are linked to a detectable label. As noted above, a number of exemplary labels are known in the art, and all such labels can be used with the present invention. These kits may include the antibody-label conjugate, either in fully conjugated form, in the form of an intermediate, or as a separate part bound by the user of the kit.

The imaging kit preferably includes a VEGFR2-blocking anti-VEGF antibody (eg, 2C3), which is already attached to a detectable label in vivo. However, the label and the binding means can be supplied separately.

[0741] Both kits further include a regulator (whether labeled or unlabeled), such as a suitably aliquoted composition of VEGF, for preparing a standard curve for a detection assay. Including, as can be used for. The components of this kit are:
It can be packaged in either an aqueous medium or lyophilized form.

[0741] The container means of the kit will generally include at least one vial, test tube,
Including a flask, bottle, syringe, or other container means, into which the antibody or antigen may be placed, and preferably in suitable aliquots. Where a second or third binding ligand or additional component is provided, the kit will also generally include a second, third, or other additional container in which the ligand or component may be located. The kit may also include other diagnostic reagents for use in diagnosing any one or more angiogenic diseases. Preferably, a second diagnostic agent that is not based on VEGF binding is used.

The kits of the invention will also typically include a means for containing the antibody, and any other reagent containers that are sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers holding the desired vials.

The following examples are included to demonstrate preferred embodiments of the invention. The techniques disclosed in the following examples represent techniques that have been found by the present inventors to work well in the practice of the present invention, and are therefore considered to constitute preferred embodiments of the present invention. Obtaining should be recognized by those skilled in the art. However, one of ordinary skill in the art will appreciate, in light of the present disclosure, that many changes may be made in the particular embodiments disclosed and that similar or similar results may be obtained without departing from the spirit and scope of the invention. It should be recognized that

Example I Generation and Specific Features of Anti-VEGF Antibody 2C3 A. Materials and Methods 1. Immunogen Corresponds to the N-terminal 26 amino acids of human VEGF (huVEGF; SEQ ID NO: 10) Peptides and peptides corresponding to the N-terminal 25 amino acids of guinea pig VEGF (gpVEGF; SEQ ID NO: 11) were synthesized from Biopolymers Facilit.
y of the Howard Hughes Medical Insti
tute, UT Southwestern Medical Center,
Synthesized by Dallas. This peptide has the following sequence (from N to C)

[0746]

Embedded image

With the following formula:

The peptide was converted to a succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) linker (Pierce, Rockfol).
d, IL) using a cysteine at the C-terminus. A control conjugate consisting of L-cysteine linked to thyroglobulin was also prepared. The conjugate was separated from free peptide or linker by size exclusion chromatography.

Recombinant human VEGF was also used as an immunogen (S. Ramakrish
Dr. Nan, University of Minnesota, Minnea
polis, obtained from MN).

(2. Hybridomas) For production of anti-gpVEGF antibody-producing hybridomas, C57 / B1-6 mice were treated with TiterMax adjuvant (CytRX Co., Norcros).
s, GA) in gpVEGF-peptide-thyroglobulin conjugate.
For production of anti-human VEGF antibodies, BALB / c mice were titrated with TiterMax.
HuVEGF-peptide-thyroglobulin conjugate or recombinant human VEG in
F. Three days later, the final boosted splenocytes were removed from Morrow et al.
990; myeloma P3X63AG8.653 (American Type Culture), as described by
Collection, Rockville, MD) cells and were cultured.

(3. Antibody Purification) The IgG antibodies (2C3, 12D7, 3E7) were purified by ammonium sulfate precipitation and protein A chromatography by Pierce ImmunoPure.
Purified from tissue culture supernatant using the Binding / Elution buffer system (Pierce).

The IgM antibody (GV39M, 11B5, 7G3) was purified from tissue culture supernatant by 50% saturated ammonium sulfate precipitation, resuspension of the pellet in PBS (pH 7.4) and dialysis against dH ₂ O, The globulin was precipitated. This dH ₂
The O precipitate is resuspended in PBS and separated on a Sepharose S300 column (
(Pharmacia). The IgM fraction was 85-90% pure as determined by SDS-PAGE.

4. Control Antibodies Various control antibodies were used throughout these studies, including: m
Ab 4.6.1 (mouse anti-human VEGF from Genentech, Inc.)
, Ab-3 (mouse anti-human VE manufactured by Oncogene Science, Inc.)
GF), A-20 (Santa Cruz Biotechnology, In)
c. , Santa Cruz, CA, rabbit anti-human VEGF), OX7 (Dr
. A. F. Williams, MRC Cellular Immunolog
mouse anti-rat Thy1.1) from Unit, Oxford, UK, MT
SA (Dr. ES Vitetta, UT-Southwestern, Da)
Illas, TX, an irrelevant specificity of mouse myeloma IgM), 1A8 (mouse anti-mouse Flk-1; Philip E. Thorpe and coworkers), ME
CA 32 (Dr. E. Butcher, Stanford University)
ty, Stanford, CA) and TEC 11
(, Mouse anti-human endoglin; US Pat. No. 5,660,
No. 827).

5. Initial Screening For initial screening, a 96-well ELISA plate (Falco
n, Franklin Lakes, NJ) were coated with either 250 ng of VEGF peptide or VEGF-Cys-thyroglobulin conjugate and blocked with 5% caseinate hydrolyzate (Sigma, St. Louis, MO). Supernatants from the anti-gp VEGF hybridoma and the first anti-human VEGF hybridoma were screened on plates coated with this antigen by dual indirect ELISA technology (Crowt).
her, 1995).

Shows preferential reactivity with VEGF peptide-thyroglobulin, but with Cys-
Hybridomas that show no or weak reactivity with thyroglobulin were further screened by immunohistochemistry on frozen sections of tumor tissue (described below).

(6. Immunohistochemistry) Guinea pig lineage 10 hepatocellular carcinoma tumor cells (Dr. Ronald Neuman,
NIH, Bethesda, MD) was obtained from line 2 guinea pigs (NCI, B
ethesda, MD). Human tumor NCI-H358 non-small cell lung cancer (NSCLC), NCI-H460 NSCLC (both Dr.Adi
Gazdar, UT Southwestern, Dallas, TX), HT29 colon adenocarcinoma (American Type Culture Col.)
lection, and L540CY Hodgkin's lymphoma (obtained from V. Diehl, Clogne, Germany) were obtained from CB17 SCID mice (
(Charles River, Wilmington, Mass.) As xenografts.

[0757] Tumors were snap frozen in liquid nitrogen and stored at -70 ° C. Frozen samples of patient-derived tumor specimens were obtained from the National Cancer Institute.
e Cooperative Human Tissue Network (S
Southern Division, Birmingham, AL).
Immunohistochemistry was performed as described in Burrows et al. (1995).

7. ELISA Analysis Hybridoma supernatants from animals immunized with VEGF were analyzed by differential indirect EL using the following three different antigens.
Screened by ISA technology: human VEGF alone, VEGF: Flk
-1 / SEAP complex, and Flk-1 / SEAP alone. For human VEGF alone, specific ELISA plates were coated with 100 ng of VEGF.

For Flk-1 / SEAP alone, 500 ng of other ELISA plates
Flk-1 / SEAP (a soluble form of the mouse VEGF receptor (Flk-1
/ SEAP-secreting cells are available from Dr. Ihor Lemischka, Prin
ceton University, Princeton, NJ)). This Flk-1 / SEAP protein was purchased from Tessler et al.
Produced and purified as described in 94). Basically, the extracellular domain of Flk-1 (sFlk-1) is replaced with Spodoptera frugiperda (S
f9) and purified by immunoaffinity technology using a monoclonal anti-Flk-1 antibody (1A8). SFlk-1 was then biotinylated and bound on avidin-coated plates.

To prepare plates coated with VEGF: Flk-1 / SEAP complex, purified sFlk-1 is biotinylated and 2,5: 1 sFlk-1: VE
In a molar ratio of GF, binding buffer (10 mM HEPES, 150 mM NaCl,
Reaction with VEGF in 20 μg / ml bovine serum albumin and 0.1 μg / ml heparin) at 4 ° C. overnight to form a dimer. Next, this VE
The GF: sFlk-1 complex was incubated in avidin-coated wells of a 96-well microtiter plate to generate a VEGF-coated plate bound to the receptor.

Next, VEGF alone, biotinylated sFlk-1 and VEGF: sFlk-
The reactivity of the antibody with one complex was determined in a controlled study using these three antigens on avidin-coated plates. Reactivity was determined as described above for the initial screen.

A capture ELISA was also developed. In a capture ELISA, microtiter plates were coated overnight at 4 ° C. with 100 ng of the indicated antibody. Wells are washed and blocked as described above, then various concentrations of biotinylated VE
Incubated with GF or VEGF: sFlk-1-biotin. 1:
Streptavidin conjugated to peroxidase (K.K.
Irkegaard & Perry Laboratories, Inc. )
Was used as the second layer and developed.

[0764] Competitive ELISA studies were performed according to the manufacturer's instructions (EZ-Link Activat).
ed Peroxidase, Pierce) by first labeling the antibody with peroxidase. 12D7, 3E7, 2C3 and 7G
The antigen used in the competition study with 3 was VEGF-biotin captured by avidin on an ELISA plate. Approximately 0.5-2.0 μg / ml of peroxidase-labeled test antibody was added to buffer alone, irrelevant IgG, or 10-10
Incubated on plates in the presence of a 0-fold excess of any of the other anti-VEGF competitor antibodies.

The binding of the labeled antibody was determined using 3,3′5,5′-tetramethylbenzidine (TMB
) Substrates (Kirkegaard and Perry Laboratories,
Inc) was evaluated. The reaction was stopped after 15 minutes with 1 MH ₃ PO ₄ and read spectrophotometrically at 450 nM. The assay was performed at least twice in triplicate for each combination of labeled and competitor antibodies. The two antibodies cross-block more than 80% of each other's binding.
) Was considered to be in the same epitope group.

GV39M and 11B5 did not retain binding activity after peroxidase labeling, but were resistant to biotinylation. GV39M and 11B5 were biotinylated and captured by anti-Flk-1 antibody (1A8) or ELISA
Tested against VEGF: sFlk-1 coated directly on A-plate.

8. Western Blot Analysis Purified recombinant VEGF in the presence of 5% fetal calf serum was separated by 12% SDS-PAGE under reducing and non-reducing conditions and transferred to nitrocellulose. This nitrocellulose membrane was applied to Sea-Block PP82-41 (Eas
t Coast Biologics, Berwick, ME) and blocked using a mini-blotter apparatus (Immun
optics, Cambridge, Mass.) was used to probe the primary antibody. After incubation with the appropriate peroxidase-conjugated secondary antibody, ECL
The film was developed by enhanced chemiluminescence.

B. Results (1.2C3 has unique epitope specificity) Table 1 shows the classes / subclasses of different anti-VEGF antibodies, the VE they recognize.
Epitope group on GF, and VEGF or VEGF: receptor (V
Summarizes information about preferential binding to the EGF: Flk-1) complex. In all cases, these antibodies bound equally well to VEGF121 and VEGF165 with essentially the same results. These results below are for VEGF165 unless otherwise specified.

[0768]

[Table 6]

[0769] 1. Epitope groups were determined by competitive ELISA. 2. Mice were treated with the N-terminal 26 amino acids of human VEGF (11B5, 3E7, 7G3
And 12D7), immunized with a synthetic peptide corresponding to either the N-terminal 25 amino acids of guinea pig VEGF (GV39M) or full length recombinant human VEGF (2C3). 3. Antibodies were tested for reactivity with VEGF alone or VEGF conjugated with sFlk-1
Indirect ELISA and capture E for reactivity with (VEGF: Flk-1)
Screened in LISA. 4. A4.6.1 has a well-defined epitope, which differs from epitope group 4 which is recognized by 2C3. The A4.6.1 study was conducted by Kim
Et al., 1992; Wiesmann et al., 1997; Muller et al., 1998; and Keyt, 1996, each of which is incorporated herein by reference.

Biotinylated or Peroxidase-Labeled Test Antibodies, and 10-10
Competition binding studies using a 0-fold excess of unlabeled competing antibody showed that 2C3 binds to a unique epitope. These studies indicate that GV39M and 11B5 cross-block each other's binding to VEGF: Flk-1 and that 3E7
And 7G3 were first shown to cross-block each other's binding to VEGF-biotin captured on avidin. GV39M and 11B5,
Epitope group 1 was arbitrarily assigned, while 3E7 and 7G3 were assigned to epitope group 2.

2C3 and the remaining antibody 12D7 did not significantly interfere with the binding of each other to VEGF or VEGF: receptor, or the binding of the remaining antibodies to them. 12D7 was assigned to epitope group 3 and 2C3 was assigned to epitope group 4 (Table 1).

[0772] As tabulated above, 2C3 recognizes an epitope that is different from the epitope for antibody A4.6.1. Our competition studies show 2C3 and A4.6.
. 1 indicated that it was not cross-reactive. The epitope recognized by A4.6.1 is also a well-defined and contiguous epitope centered around amino acids 89-94 (Kim et al., 1992; Wiesmann et al., 199).
7; Muller et al., 1998; Keyt et al., 1996, each of which is incorporated herein by reference. There are also a number of known differences between 2C3 and A4.6.1 (see below).

(2.2C3 Binds Free VEGF) In the ability of the antibody to bind to free and complex forms of soluble VEGF,
There are significant differences (Table 2). These studies provide further evidence for the unique nature of 2C3. Table 2 shows that GV39M and 11B5 exhibited strong selectivity for the VEGF: receptor complex, with half-maximal binding (half-maximal).
binding) is 400n for free VEGF in solution, respectively.
M and 800 nM, as compared to VEGF: Flk-1.
Reached at 5 nM and 2 nM.

[0774] In contrast, 2C3 and 12D7 exhibited selectivity for free VEGF,
The half-maximal binding was 150 for the VEGF: Flk-1 complex, respectively.
Reached at 1 nM and 20 nM, respectively, compared to nM and 250 nM. However, 2C3 is localized to the tumor vasculature and stroma after in vivo injection (see below).

3E7 binds equally to free VEGF and the VEGF: Flk-1 complex,
The half-maximal binding is reached at 1 nM for both.

[0776]

[Table 7]

[0777] 1. Half-maximal binding values were determined by titrating biotinylated VEGF, and biotinylated sFlk-1 complexed with VEGF, on wells coated with the indicated antibodies in triplicate, followed by color development with peroxidase-labeled avidin. Measured. Ratios greater than 2.1.0 indicate selectivity of the antibody for the conjugate (VEGF: Flk-1), while ratios less than 1.0 indicate selectivity for VEGF. 3. The control antibodies used include the following: 1A8 (mouse anti-Flk-1), Mab 4.6.1 (mouse anti-human VEG from Genentech)
F), and irrelevant IgM as a negative control. 4. NR = No reaction detected.

(3.2C3 Recognizes Non-Conformation Dependent Epitope) Western blot analysis shows that 12D7, 2C3 and 7G3 react with denatured VEGF121 and VEGF165 under reducing and non-reducing conditions. Show. Thus, these antibodies are non-conformation dependent (non-c
It appears to recognize an epitope of informationally-dependent.

[0779] In contrast, GV39M, 11B5 and 3E7 showed VV on Western blots.
Did not react with EGF. This is probably because they recognize an epitope on the N-terminus of VEGF that is conformation-dependent and deforms under denaturing conditions.

[0780] Western blot analysis of anti-VEGF antibodies was performed using SDS-PAGE on a 12% gel.
Using E, VEGF165 was isolated in the presence of 5% FCS and analyzed by a standard Western blotting protocol using ECL detection. Primary antibodies were incubated with the nitrocellulose membrane using a multi-lane mini blotter device. Control antibodies included: 1 μg / ml Ab-3 (VEG from Oncogene Science).
F-specific monoclonal IgG), 5 μg / ml A-20 (Santa
Cruz Biotechnology, Inc. Rabbit anti-VEGF antibody)
, And IgG of irrelevant specificity of 10 μg / ml.

A representative Western blot for the different antibodies, containing 5 μg / ml of 2C3, showed the dimeric VEGF as a large band of approximately 42 kd. About 13
A 0 kd multimeric VEGF was also revealed with 12D7, 7G3 and positive control antibodies.

4. Tumor Immunohistochemistry Tumors tested by immunohistochemistry include various types of human tumors from cancer patients, various types of implantable human tumor xenografts grown in mice, Guinea pig lineage 10 tumors grown in guinea pigs, and mouse 3LL grown in mice
Tumor (see description to Table 3 for details).

[0778] The immunohistochemical reactivity of 3E7, GV39M and 11B5 to NCI-H358 human NSCLC xenografts was measured and control antibodies (IgG of irrelevant specificity; A20 (rabbit anti-VEGF antibody); and MECA 32 (
Rat anti-mouse endothelial cell antibody). NCs grown in SCID mice
8 μm frozen sections of I-H358 human NSCLC were stained using indirect immunoperoxidase technology and counterstained with hematoxylin.

GV39M and 11B5, which recognize epitope group 1 on VEGF, strongly stain vascular endothelial cells and moderately stain connective tissue around blood vessels in all tumors tested Was decided. Epitope group 1 antibodies differ in their reactivity with tumor cells, with GV39M reacting weakly with tumor cells, while 11B5 reacted more strongly. MECA
About 8 of endothelial cells stained by T.32 (mouse) or TEC 11 (human)
0% was also stained by GV39M and 11B5.

[0785] 3E7 and 7G3 (these recognize VEGF epitope group 2)
Showed reactivity with vascular endothelial cells, connective tissue, and tumor cells in all tumors tested (Table 3). In particular, when the antibody is applied at low concentrations (1-2 μg / ml), the intensity of endothelial cell staining is typically stronger than tumor cell staining or connective tissue staining, with marked selectivity for vascular endothelium. Significant increase was seen.

[0786]

[Table 8]

[0787] Immunohistochemical analysis was performed on acetone-fixed frozen sections of tumor tissue by standard immunohistochemical techniques. These sections were examined microscopically and scored for reactivity as follows:-, negative; +/-, very weak; 1+, weak; 2+, moderate; 3+, strong; 4+, very strong. 1.2C3 and 12D7 were applied at 20 μg / ml; all other antibodies were
Applied at 〜１０10 μg / ml. 2. Definition of reactivity: EC = endothelium; CT = connective tissue; TC = tumor cells; NR = no response. 3. Human tumor xenografts tested; NCI-H358 NSCLC, NCI-H
460 NSCLC, HT29 colon adenocarcinoma, L540 Hodgkin lymphoma. 4. Human tumors tested; soft tissue sarcoma, Hodgkin lymphoma, kidney cancer, breast cancer, parotid gland cancer, colon cancer, lung cancer and endothelial cancer. 5. Reactivity with guinea pig VEGF in line 10 guinea pig tumor sections. 6. Reactivity with mouse VEGF in mouse 3LL Lewis lung cancer tumor sections.

[0788] 12D7 and 2C3 did not stain frozen sections of any tumor tissue. This is probably because fixation of the tissue with acetone disrupted antibody binding. However, 2C3 localized to tumor tissue after in vivo injection (see below).

GV39M, 11B5, 3E7 and 7G3 reacted with rodent vasculature on cryosections of guinea pig line 10 tumors grown in guinea pigs and mouse 3LL tumors grown in mice. GV39M, 11B5 and 7G3 reacted strongly with guinea pig and mouse tumor vasculature as well as with human vasculature in human tumor specimens. 3E7 stained mouse 3LL tumors at a lower intensity than staining guinea pig or human tumor sections. This is because 3E7 is mouse VEG
Suggests having lower affinity for F. These results are consistent with analysis by indirect ELISA, which shows that all antibodies except 2C3 react with mouse VEGF.

Example II 2C3 Inhibits Endothelial Cell Migration A. Materials and Methods Endothelial Cell Proliferation Assay Adult bovine aortic endothelial (ABAE) cells were cultured at 1500 cells / well in 96-well tissue Plated in culture plates and grown in the presence of 0.5 nM human VEGF with the addition of various sample and control antibodies. Control wells contained medium with or without VEGF.

[0790] Four days after the incubation, the cells were treated with MTC (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-
Sulfophenyl) -2H-tetrazolium, inner salt
Quantified by conversion assay. Here, MTS conversion to formazan is proportional to cell number and can be tracked by absorbance at 490 nM (Cell Ti
ter 96 Aqueous One Solution Cell Pro
Licensing Assay, Promega, Madison, WI)
. This assay was performed according to the manufacturer's instructions.

[0792] Cell proliferation was estimated by subtracting MTS conversion in cultures without VEGF addition. The results were expressed as a percentage of MTS conversion in control cultures to which only VEGF was added (Buttke et al., 1993).

B. Results Inhibition of VEGF-Mediated Endothelial Cell Proliferation The IgG antibodies 3E7 and 12D7, which recognize epitope groups 1-3 on VEGF, did not inhibit VEGF-mediated proliferation of ABAE cells (FIG. 1). ). This suggests that these antibodies are non-blocking anti-VEGF antibodies whose epitopes are not involved in VEGF: KDR interaction.
IgG antibody GV39M, which also recognizes epitope groups 1-3 on VEGF
Also does not inhibit VEGF-mediated proliferation of ABAE cells and unblocked anti-VE
GF antibody.

In contrast, 2C3 against epitope 4 on VEGF, and the reference neutralizing anti-V
The EGF antibody, Mab 4.6.1, was used at 3 nM and 1 nM, respectively, for VEGF
Inhibited mediated ABAE proliferation by 50% (FIG. 1). This indicates that 2C3 can neutralize the mitogenic activity of VEGF.

The definition of a 2C3 as a blocking Mab distinguishes 2C3 from a range of other antibodies in addition to GV39M, 3E7 and 12D7. Various anti-VEGF antibodies (eg, Ab-3) are known to be non-blocking monoclonals, and are distinct from 2C3.

One of the IgM antibodies, 11B5, was toxic to ABAE cells at concentrations higher than 8 nM. Cells detached within 30 minutes and 24 hours incorporated trypan blue dye. This effect appears to be cell type specific. HUVEC, porcine aortic endothelial cells, and bEND. 3 cells are 40n
Even M was not affected by 11B5. As occurs in the absence of the addition of VEGF and in the presence of basic fibroblast growth factor (bFGF),
The toxic effects of 1B5 appear to be growth factor independent.

Example III 2C3 Localizes specifically to Tumors In Vivo A. Materials and Methods In Vivo Localization to Human Tumor Xenografts It was grown subcutaneously in immunocompromised mice to about 1 cm ³ (NCI-H358 NSCLC in nu / nu mice and HT29 colon adenocarcinoma in SCID mice). 100 μg of unlabeled antibody for studies using SCID mice or 100 μg of biotinylated antibody for studies using nude mice were injected intravenously via the tail vein. Twenty-four hours later, mice were anesthetized, perfused with PBS, and tumors and organs, including heart, lung, liver, kidney, intestine and spleen, were collected and snap frozen in liquid nitrogen.

[0798] Tumors and organs from each mouse were sectioned on a cryostat and stained for antibodies immunohistochemically as described above. However, a section derived from a nude mouse was obtained by using a peroxidase-labeled streptavidin-biotin complex (Dako, Ca
(Pinteria, Calif.) and sections from SCID mice were treated with two peroxidase-conjugated secondary antibodies (goat anti-mouse IgG + Ig).
M, followed by rabbit anti-goat IgG).

B. Results In Vivo Localization in Tumor-Bearing Mice The in vivo localization of 2C3, 3E7 and GV39M in human tumor xenografts was determined. 100 μg of biotinylated 2C3 or isotype matched control IgG was injected i.v. into nu / nu mice bearing NCI-H358 human NSCLC. v. Injected. 100 μg of GV39M and 3E7 or isotype matched control IgG were injected into SCID mice bearing HT29 human colon adenocarcinoma. Twenty-four hours later, mice were sacrificed, exsanguinated, and tumors and tissues removed. Cryosections of tumors and tissues were analyzed immunohistochemically to determine antibody binding and distribution (Table 4).

(Table 4. Tissue distribution of anti-VEGF antibody in tumor-bearing mice)

[0801]

[Table 9]

The immunohistochemical analysis was performed on cryosections of acetone-fixed tissues (including tumors) from tumor-bearing mice that received 100 mg of the indicated antibody intravenously 24 hours prior to sacrifice. Ran with The sections were examined microscopically and scored for specific reactivity as follows:-, negative; +/-, very weak;
+, Weak; 2+, moderate; 3+, strong; 4+, very strong. 1. GV39M specifically bound to endothelial or mesangial cells in the glomeruli of the kidney. 2.3E7 and GV39M specifically bound to tumor vascular endothelium while 2
C3 specifically bound to tumor stroma. 3. Control = IgM of irrelevant specificity.

[0803] 3E7 localized specifically to vascular endothelium in tumors. About 70% MECA
32 positive vessels were stained by 3E7 injected in vivo. Larger vessels giving the microvasculature were 3E7 positive. Small microvessels were also positive for 3E7, both in stromal tracks and in tumor nests. The intensity of staining with 3E7 increased in and around areas of focal necrosis. Extravascular antibodies were evident in areas of tumor necrosis, but there was little evidence of extravascular staining in healthy areas of the tumor. The vascular endothelium in all normal tissues tested (including kidneys)
No staining by 3E7.

GV39M also localized specifically to tumor vascular endothelium. Approximately 80% of the MECA 32 positive vessels in the tumor were stained by GV39M. GV39M positive vessels were equally distributed throughout the tumor (including large vessels but also small capillaries). Similar to 3E7, the staining intensity of GV39M positive vessels increased in necrotic areas of tumor foci. But 3
Unlike E7, renal glomerular endothelial cells or mesenchymal cells were also stained. Since control IgM of unrelated specificity does not result in glomerular staining,
Glomerular staining with V39M appears to be antigen-specific. Vascular endothelium in tissues other than kidney was not stained by GV39M.

Biotinylated 2C3 resulted in strong staining of the connective tissue surrounding the vasculature of H358 human NSCLC tumors after intravenous injection. Large tracks of stromal tissue connecting nests of tumor cells were stained by 2C3, where the strongest localization was observed in the largest track of stromal cells. In these areas, it was not possible to distinguish the vascular endothelium from the surrounding connective tissue. However, endothelial cells in blood vessels that were not surrounded by stroma (eg, endothelial cells in blood vessels running through the nest of the tumor tissue itself) were stained. In any normal tissue tested,
There was no detectable staining with 2C3.

In the HT29 human tumor model, 2C3 was also strongly localized to connective tissue, but most intense staining was observed in necrotic areas of the tumor.

Example IV 2C3 Inhibits VEGF Binding to VEGFR2, but Not VEGFR
(A. Materials and Methods) 1. Cell Lines and Antibodies Porcine aortic endothelial (PAE) cells transfected with either VEGFR1 (PAE / FLT) or VEGFR2 (PAE / KDR) , Dr. Joh
Annes Waltenberger (Ulm, Germany), which was prepared as described in Waltenberger et al. (1994, specifically incorporated herein by reference) and 5% FCS, L-glutamine , Penicillin, and streptomycin (GPS). bEND3 cells were obtained from Dr. Werner Risau (Ba
d Nauheim, Germany) and grown in DMEM medium containing 5% FCS and GPS. NCI-H358 NSCLC (D
r. Adi Gazdar, UT-Southwestern, Dallas,
TX), A673 human rhabdomyosarcoma, and HT1080 human fibrosarcoma (both from the American Type Culture Collection)
on) were grown in DMEM medium containing 10% FCS and GPS.

[0808] 2C3 and 3E7 (anti-VEGF monoclonal antibody), and 1A8 (monoclonal anti-Flk-1 antibody), and T014 (polyclonal anti-Flk-1)
Antibodies) are described in Example I as described above and in Brekken et al. (1998) and Huang et al. (1998), each of which is specifically incorporated herein by reference. A4.6.1 (mouse anti-human VEGF monoclonal antibody) was obtained from Dr. A. Obtained from Jin Kim (Genentech Inc., CA) and described previously (Kim et al., 1992; specifically incorporated herein by reference). The negative control antibodies used were OX7, a mouse anti-rat Thr1.1 antibody (Bukovsky et al., 1983) (Dr. A.
F. Williams (MRC Cellular Immunology U
nit, Oxford, UK) and C44, a mouse anti-colchicine antibody (Rouan et al., 1990, obtained from ATCC).

(2. ELISA analysis) The extracellular domain of VEGFR1 (Flt-1 / Fc, R & D Systems
, Minneapolis) or VEGFR2 (sFlk-1-biotin)
Were coated directly on the wells of a microtiter plate or captured by wells coated with NeutrAvidin (Pierce, Rockford, IL), respectively. VEGF at a concentration of 1 nM (40 ng / ml)
Was incubated in wells with or without 100-1000 nM (15 μg-150 μg / ml) of control or test antibody. Then, the wells were treated with a 1 μg / ml rabbit anti-VEGF antibody (A-20, Santant).
a Cruz Biotechnology, Santa Cruz, CA).

The reaction was performed using a peroxidase-labeled goat anti-rabbit antibody (Dako, Carpi).
color, and addition of 3,3'5,5'-tetramethylbenzidine (TMB) substrate (Kirkegaard and Perr).
y Laboratories, Inc. ) Was visualized. After 15 minutes, the reaction was stopped with 1 MH ₃ PO ₄ and read spectrophotometrically at 450 nm.

The assay was also performed by coating the wells of a microtiter plate with either control IgG or test IgG. The wells were then incubated with VEGF: Flt-1 / Fc or VEGF: sFlk-1-biotin and, respectively, a peroxidase-labeled goat anti-human Fc (
Kirkegaard and Perry Laboratories, In
c. ) Or peroxidase labeled streptavidin were developed and visualized as described above.

3. Co-Precipitation Assay Forty ng of VEGF was combined with either 2C3 (20 μg) or A4.6.1 (10 and 1 μg) F (ab ′) _{2 for} 30 minutes in binding buffer (1 mM).
DM with CaCl ₂ , 0.1 mM CuSO ₄ , and 0.5% tryptone
EM). 200 ng of soluble VEGFR1 (Flt
−1 / Fc) or VEGFR2 (KDR / Fc, R & D Systems, M
(Inneapolis, MN) was added in a total volume of 50 μl and incubated for 2 hours. The receptor / Fc construct was used for protein A-sepha
Precipitated using ose beads, and the resulting precipitate was washed four times with binding buffer.

[0813] Reduced sample buffer was added to the pellet and supernatant of each reaction and both were added to 12%
Analyzed by SDS-PAGE and transferred to PVDF membrane. The membrane was then probed with 12D7 (1.0 μg / ml) (mouse anti-VEGF antibody) and peroxidase-labeled goat mouse IgG (Kirk
egard & Perry Laboratories, Inc. ) Followed by Super Signal chemilumines
Color was developed by Cence Substrate (Pierce, Rockford, IL). The soluble receptor / Fc construct is also a peroxidase-conjugated goat anti-human Fc (Kirkegaard & Perry Labora).
tories, Inc. ).

(B. Results) (1.2C3 blocks binding of VEGF to VEGFR2 but not VEGFR1 in ELISA) Anti-VEGF antibody 2C3 demonstrates that VEGF is
Blocks binding to R2 (KDR / Flk-1), but binds to VEGFR1 (
Binding to FLT-1) was not blocked. VE binding to VEGFR2-coated wells in the presence of 100-fold and 1000-fold molar excess of 2C3
The amount of GF was reduced to 26% and 19%, respectively, of the amount bound in the absence of 2C3 (FIG. 2). In contrast, 100-fold and 1000-fold molar excess of 2C3
The amount of VEGF bound to VEGFR1 coated wells in the presence of was 92% and 105% of the amount bound in the absence of 2C3, respectively (FIG. 2).

The amount of VEGF that binds to VEGFR1 or VEGFR2 is 100-100
It was unaffected by the presence of a 0-fold excess of non-blocking monoclonal anti-VEGF antibody 3E7 or control IgG of unrelated specificity (FIG. 2). A
4.6.1 is the VEGFR2 (KDR / Flk-1) and VEGFR1 (FLT-
VEGF binding to both of 1) was blocked.

(2.2C3 blocks VEGF binding to VEGFR2 but not VEGFR1 in solution) 2C3 is VEGF VEGFR1 / Fc or VEGFR2 / F in solution
The ability to block binding to c was evaluated in a co-precipitation assay. 40ng
VEGF was linked to VEGFR2 (KDR / Fc) with or without the extracellular domain of VEGFR1 linked to the Fc portion (Flt-1 / Fc) or 2C3 or 4.6.1 F (ab ′) ₂ ) Was incubated with 200 ng. Receptor / Fc constructs were precipitated by incubation with Protein A sepharose beads. The precipitate is washed, resuspended in reducing sample buffer and separated by 12% SDS-PAGE, and
Transferred to VDF. Block membrane with P82 and 12D7 (1 μg
/ Ml) probed with mouse anti-VEGF antibody and developed under standard chemiluminescent conditions. VEGF monomers and dimers with F (ab ') ₂ were detected.

VEGF mixed with either VEGFR1 / Fc or VEGFR2 / Fc
Was co-precipitated with Protein A sepharose. This means that V
4 shows that EGF binds to both receptors. The addition of 2C3 F (ab ') ₂ blocked the binding of VEGF to VEGFR2 / Fc, but the VEGFR1
Binding to / Fc was not blocked. In contrast, 4.6.1 F (ab ') ₂
Blocked VEGF binding to both VEGFR2 / Fc and VEGFR1 / Fc. This result shows that 2C3 inhibits VEGF binding to VEGFR2 but not VEGFR1, whereas the 4.6.1 antibody shows VEGF binding to both VEGFR2 and VEGFR1. Support for inhibition.

Example V 2C3 Blocks VEGF-Induced VEGFR2 Phosphorylation A. Materials and Methods Immunoprecipitation and Western Blot Analysis PAE / KDR, PAE / FLT, and bEND . 3 cells were grown to 80-90% confluence in 100 mm tissue dishes in medium containing 5% serum. The cells were then serum starved for 24 hours in medium containing 0.1% serum. After a 30 minute pretreatment with 100 nM sodium orthovanadate in PBS, cells were incubated with 5 nM (200 ng / ml) VEGF 165, 5 nM (200 ng / ml) in the presence or absence of a control or test antibody for an additional 15 minutes.
nM (100 ng / ml) bFGF (R & D Systems, Minneap
olis, MN), or A673 tumor conditioned medium.

[0839] The cells were then treated with 10 mM EDTA, 2 mM sodium fluoride and 2 mM.
Wash with ice-cold PBS containing M sodium orthovanadate and lysis buffer (50 mM Tris, 150 mM NaCI, 1% Nonidet)
P-40, 0.25% deoxycholic acid, 0.1% CHAPS, 5 mM
EDTA, 1.5 mM MgCl _2, 2mM sodium fluoride, sodium 2mM orthovanadate, 10% glycerol, and protease inhibitors (Complete Protease Inhibitor Cockt
ail tabletss, Boeringer Mannheim). The lysate was clarified by centrifugation and the resulting supernatant was used for immunoprecipitation.

VEGFR1 and VEGFR2 were prepared by lysing cell lysates with 5 μg of chicken anti-FLT-1 N-terminus (Upstate Biotechnology, La.
(K. Placid, NY) or 10 μg T014 (affinity-purified anti-Flk-1) by incubating overnight at 4 ° C. Reactions using chicken anti-FLT-1 antibody were subsequently followed by cross-linked goat anti-chicken antibody (Kirkegaard and Perry Labora).
tories, Inc. ) At 4 ° C. for 1 hour. Then
This immune complex is precipitated using protein A / G sepharose,
Washed multiple times with 10% lysis buffer (with the addition of protease inhibitors) in PBS-Tween (0.2%) and boiled in SDS sample buffer containing 100 mM β-mercaptoethanol and 8 M urea.

[0821] The samples were then separated by SDS-PAGE and transferred to a PVDF membrane. This membrane is referred to as PP81 (East Coast Bio
Logics, Berwick, ME) for 30-60 minutes and 0.5 μg / ml of 4G10 (Upstate B) for phosphotyrosine residues.
(Technology, Lake Placid, NY) at 4 ° C. overnight. Peroxidase labeled rabbit anti-mouse IgG (Dako
, Carpinteria, CA) after incubation with Super.
Signal chemiluminesence substrate (P
ierce, Rockford, IL) to develop the PVDF membrane. Next, the PVDF membrane was washed with ImmunoPure Elution.
Strip with a buffer (Pierce, Rockford, IL) at 55 ° C. for 30 minutes and add 0.5 μg / ml chicken anti-FLT-1 or 1.0 μg
/ Ml T014 was reprobed for receptor levels and developed after incubation with an appropriate peroxidase-conjugated secondary antibody as described above.

B. Results Blocking of VEGF-Induced Phosphorylation In these studies, PAE / KDR cells were isolated from PBS, bFGF (5 nM,
100 ng / ml), VEGF165 (5 nM, 210 ng / ml), A67
3 conditioned medium (CM), specific antibodies (CNTL, 2C3, 3E7, A4.6.1,
CM combined with 100 nM, 15 g / ml separately, or T014 alone (
(100 nM, 15 μg / ml) for 15 minutes. PAE / FLT cells were also transferred to PBS, VEGF165 (5 nM, 210 ng / ml), A673 conditioned medium (
CM), specific antibodies (2C3, 3E7, A4.6.1, 100 nM separately, 15
g / ml) or T014 alone (100 nM, 15 μg / ml).
ml) for 15 minutes. The cells are then incubated in lysis buffer and the receptors immunoprecipitated, separated by SDS-PAGE under reducing conditions, transferred to a PVDF membrane, and treated with 4G50 (0.5 μg / ml) (
(Mouse anti-phosphotyrosine antibody) and developed under standard chemiluminescent conditions. The membrane was then stripped and reprobed with immunoprecipitated IgG to determine the level of receptor protein in each lane.

The results show that 2C3, together with A4.6.1 (control neutralizing anti-VEGF antibody), blocks VEGF-induced phosphorylation of VEGFR2 in PAE / KDR cells. This means that both 2C3 and A4.6.1.
Consistent with previous experiments demonstrating blocking F-mediated endothelial cell proliferation (Example II; Brekken et al., 1998). Western blots of VEGFR2 in immunoprecipitates were performed to demonstrate the amount of VEGFR2 protein in each lane. 3E7 (which, NH ₂ of VEGF - recognizing the terminal epitopes) does not block VEGF-induced phosphorylation of VEGFR2, and unrelated specificity of control IgG did not block.

The effect of 2C3 on VEGF-induced phosphorylation of VEGFR1 is not clear. As other investigators have shown, VEGF in PAE / FLT cells
EGF-induced phosphorylation of R1 is difficult to demonstrate, probably due to the low endogenous kinase activity of VEGFR1 (De Vries et al., 1992;
Waltenberger et al., 1994; Davis-Smyth et al., 1996.
Landgren et al., 1998).

Example VI 2C3 Inhibits VEGF-Induced Permeability A. Materials and Methods Miles Permeability Assay The following protocol is described by Murohara et al. (1998; herein). Which are specifically incorporated by reference). In short, 400
~ 450 g of male IAF hairless guinea pigs (Charles River, Wil
(Mington, MA) was anesthetized and then injected intravenously through the ear vein with 0.5 ml of 0.5% Evan's blue dye in sterile PBS. Twenty minutes later, 20 ng of VEGF was injected intradermally (id) in the presence or absence of a control or test antibody. Thirty minutes after intradermal injection, the resulting blue spot on the back of the guinea pig was photographed and measured using a caliper.

B. Results (2C3 blocks VEGF-induced permeability) To study the effect of 2C3 on VEGF-induced permeability, 400-
IAF hairless guinea pigs (Hartley strain) of 450 g size were anesthetized and then passed through the ear vein with 0.5 ml of 0.5% E in sterile PBS.
van's blue dye was injected intravenously. After 20 minutes, 25 ng of VEGF was injected intradermally (id) in the presence or absence of control or test antibodies.
Injected. Thirty minutes after intradermal injection, the resulting blue spot on the back of the guinea pig was photographed and measured using a caliper.

Using this Miles permeability assay, 2C3, which blocks VEGF from activating VEGFR2, was found to inhibit VEGF-induced permeability in guinea pigs. This effect is 10-fold molar excess over VEGF, 1
A 00-fold molar excess, or a 1000-fold molar excess, was evident with 2C3.
A4.6.1 that blocks VEGF from activating both VEGFR1 and VEGFR2, which blocks VEGF from activating both VEGFR1 and VEGFR2, is VEGF-induced in a 10-fold molar excess. Permeation was blocked (this study and Kim et al., 1992). 3E7 and control IgG, which do not block the VEGF: VEGFR2 interaction, were also expressed in guinea pig M
It did not block VEGF-induced permeability in the iles permeability assay.

[0827] These results suggest that endothelial permeability mediated by VEGF is mediated, at least in part, through VEGFR2 activation. These results are consistent with the results of other investigators indicating that tyrosine kinase activity of VEGFR2 was required for VEGF-induced permeability (Murohara et al., 1998; Joukov et al., 1998; Ogawa et al.). , 1998).

(Example VII) (Anti-tumor effect of 2C3) (A. Materials and methods) (1. In vitro tumor growth inhibition) Nu / nu mice were cultured on day 0 in 1 × 10 ⁷ NCI-H358 NSCLC cells. Alternatively, either 5 × 10 ⁶ A673 rhabdomyosarcoma cells were injected subcutaneously. Mice received an intraperitoneal injection of 2C3 (1, 10, or 100 μg as indicated or control) on day 1 and twice per week thereafter. Then
Tumors were measured twice a week over a period of approximately 6 weeks for mice bearing NCI-H358 and 4 weeks for mice bearing A673. Tumor volume was measured according to the formula: volume = L × W × H, where L = length, W = width, H = height.

[0830] (2 Vivo Tumor therapy) 200 to 400 mm ³ in size subcutaneous NCI-H358 tumors or HT108
Male nu / nu mice with 0 fibrosarcoma were injected intraperitoneally with test or control antibodies. Mice bearing NCI-H358 were dosed three times a week for the first week, and twice weekly for the second and third weeks, 100 per injection.
Treated with μg of antibody. The mice were then injected daily for 5 days with 5 injections per injection.
Switched to 0 μg. HT1080-bearing mice were treated with 100 μg of the indicated antibody or saline every other day for the entire duration of the study. In both studies, mice whose tumors once they reach a size of 2500 mm ^3, or earlier in the case where the tumor has started making ulcers were sacrificed.

B. Results 1. 1. Inhibition of 2C3 Growth of Newly Transplanted Human Tumor Xenografts 2C3 was expressed in a dose-dependent manner in NCI-H358 NS in nu / nu mice.
Inhibits the in vivo growth of both CLC and A673 rhabdomyosarcoma (FIGS. 3A and 3B). Mice injected subcutaneously with tumor cells were given i. p. 100 μg of 2C3 given in 1. inhibited the growth of both human tumor types one day earlier. 2
The final tumor volume in C3 recipients was about 150 mm ³ in both tumor lines as compared to about 1000 mm ³ in control recipients.

[0832] Treatment with 2C3 at either 10 μg or 1 μg twice per week was not very effective in preventing tumor growth. However, both lower doses of 2C3 slowed the growth of A673 tumors to a similar extent when compared to untreated mice. The tumor growth delay caused by a 10 μg dose of 2C3 was
Less pronounced in the H358 tumor model. The differences between these two tumor models and their response to inhibition of VEGFR2 activity by 2C3 are associated with aggression of the two types of tumors in vivo. NCI-H358 is A
673 grows even more slowly in vivo than does the low dose of 2C
A673 tumors appear to be less sensitive to
Proliferates faster and more aggressively, and appears more sensitive to C3.

[0838] 3E7, which binds to VEGF but does not block its activity,
It did not affect the growth of CI-H358 tumors. However, twice a week, 10
3E7 given at a dose of 0 μg stimulated the growth of A673 tumors (FIG. 3B). This suggests that it increases the effect of VEGF signaling in tumors.

Treatment of established human tumor xenografts with 2.2C3 Subcutaneous NCI-H358 NSCs grown to a size of about 300-450 mm ³
In mice bearing LC tumors, irrelevant specificities of 2C3, A4.6.1, 3E7,
Or IgG as i. p. Injected (FIG. 4). The dose is 50-10 every 3-5 days
It was 0 μg. A6.4.1 was used as a positive control. because,
Because it has been shown by others to block VEGF activity, which causes inhibition of tumor growth in vitro (Kim et al., 1993; Mesia
no et al., 1998). In addition to measuring the average tumor volume (FIG. 4), photographs of mice from each treatment group were also taken to show differences and appearance in tumor size at the end of the study.

[0832] Treatment with either 2C3 or A4.6.1 led to a slow regression of the tumor over the duration of the study. The mean tumor volume at the end of the study was 3 times the starting mean tumor volume.
It was 0% (2C3) and 35% (4.6.1) (FIG. 4). However, these results are complicated by the fact that a spontaneous regression in tumor growth was observed in the control group of mice between 40 and 60 days after injection of tumor cells. Results by day 40 show that 2C3 and A4 before spontaneous regression in growth
. 6 shows that treatment with 6.1 prevents tumor growth.

FIG. 5A shows a further study in which mice bearing NCI H358 were treated with 100 μg of either 2C3 or 3E7 chronically. In this study, spontaneous regression was less obvious. The mean tumor volume of 2C3-treated mice at the start of treatment was 480 mm ³ , and the mean tumor volume after approximately 14 weeks of treatment was reduced to 84 mm ³ (（80% reduction in volume). 3E7 treated mice were treated starting at an average tumor volume of 428mm ^3, and after about 14 weeks increased to a volume of 1326mm ³ (an increase of 300% by volume).

FIG. 5B shows a tumor growth curve of a mouse with HT1080, a human fibrosarcoma. The mice were treated with 100 μg of 2C3, 3E7, or control IgG, or saline every two days. 2C3, which hindered tumor growth, was continued for as long as treatment. Mice treated with 3E7, control IgG, or saline had tumors that progressively grew to the required size for mice to be sacrificed less than 4 weeks after tumor cell injection.

Example VIII (2C3 is distinguished from A4.6.1) There are numerous differences between 2C3 and A4.6.1 (eg, Table 5). Antibodies recognize distinct epitopes on VEGF based on an ELISA cross-block study (Example I). Mutational and X-ray crystallographic studies have shown early that A4.6.1 binds to an epitope on VEGF concentrated around amino acids 89-94 (Muller et al., 1998).

[0839] Of particular interest, A4.6.1 blocks VEGF from binding to both VEGFR1 and VEGFR2 (Kim et al., 1992; Wiesma).
nn et al., 1997; Muller et al., 1998; Key et al., 1986)
3 is the fact that it blocks VEGF only from binding to VEGFR2 (Example IV). A4.6.1 VEGF to VEGFR2 and VEGFR1
Strong published evidence for inhibiting binding has been obtained from detailed crystallographic and structural studies (Kim et al., 1992; Wiesmann et al., 1997; Muller).
Et al., 1998; Keyt et al., 1996; each of which is incorporated herein by reference). Published data indicate that A4.6.1 inhibits VEGF binding to VEGFR2 by competing with epitopes on VEGFR that are important for binding to VEGFR2, and inhibits VEGF binding to VEGFR1. Most preferably blocks by steric hindrance (Muller et al., 1998; Keyt et al., 19).
96).

A humanized version of A4.6.1 is currently in clinical trials (Brem, 19
98; Baca et al., 1997; Presta et al., 1997; each of which is incorporated herein by reference). Macrophage / monocyte chemotaxis and VEGFR1
The other endogenous functions of VEGF mediated via are likely to be most impaired in the A4.6.1 trial. In contrast, 2C3 is predicted to be better due to its ability to specifically block VEGFR2-mediated effects. Thus, 2C3 is a potentially safer antibody, especially for long-term administration to humans. The benefits of treatment with 2C3 include the ability of the host to mount a greater anti-tumor response by allowing VEGF-induced tumor vasculature expansion, while at the same time allowing macrophages to migrate to the tumor . Also, many systemic benefits of maintaining macrophage chemotaxis and other effects mediated by VEGFR1 should not be overlooked.

[0841]

[Table 10]

Abbreviations used: IHC, immunohistochemistry; NR, unreactive; BV, blood vessels; CT,
Connective tissue. 1. References for A4.6.1 data include: Kim
Wiesmann et al., 1997; Muller et al., 1998; and Keyt et al., 1996, each of which is incorporated herein by reference. The epitope recognized by 2.2C3 on VEGF is undetermined, but A4.6. .1 is E
2. It has been shown to be distinguished from epitopes recognized through LISA cross-block studies. 3. The affinity of 2C3 for VEGF has been assessed by ELISA and surface plasmon resonance analysis. A4.6.1 only reacts with lightly fixed acetone-fixed frozen sections (Example IX) (VEGF staining in joint tissue) The relationship between angiogenesis and disease is angiogenesis It extends beyond the relationship observed in tumors. For example, the involvement of abnormal angiogenesis has been well described in arthritis. A panel of different anti-VEGF antibodies to thymidine phosphorylase has been used to stain arthritic tissue and differentiate it from matched controls. Studies using antibodies to VEGF have shown significant expression in the pannus of rheumatoid arthritis.

Example X 2C3-Endostatin Conjugate A. Cloning and Expression of Endostatin RNA was isolated from mouse liver and RT-PC using the following primers
Used as a template for the R ^TM:

[0844]

Embedded image

The obtained cDNA fragment has the DNA sequence of SEQ ID NO: 12 and has the amino acid sequence of SEQ ID NO: 13. For reference, the human endostatin amino acid sequence is SEQ ID NO: 14. The mouse cDNA fragment was transformed into the expression vector H6p.
QE60 (Qiagen) (which encodes an N-terminal 6 × histidine tag)
And then cloned into E. coli. E. coli M15 cells. E. FIG. c
When the oli cell density reached an optical density of 0.6 at 560 nM, 0.1 mM isopropylthiogalactoside (IPTG) was added to induce 6-His endostatin expression for 4 hours. Cells are collected by centrifugation and lysis buffer (B
-PER Bacterial Protein Extraction Re
agent (Pierce, Rockford, IL).

The inclusion bodies (containing 6-His tagged endostatin) were sedimented by centrifugation and buffer A (pH 8.0, 6M guanidine HCl (GuHCL), 100
mM NaH ₂ PO ₄ , 10 mM Tris, 10 mM imidazole, 10 mM
β-2 mercaptoethanol). The solution containing reduced 6-His endostatin is treated with excess 5,5'-dithio-bis-2-nitrobenzoic acid (Ell
(man's reagent) (20 mM) and loaded onto a Ni-NTA column. The column was washed with wash buffer (6 M GuHCl, 100 mM NaH ₂ PO ₄ ,
Wash with 10 mM Tris, 500 mM NaCl, pH 7.3) and 6
-His endostatin was eluted from the column with 0.2 M imidazole in wash buffer.

The eluted and insoluble 6-His endostatin was added to an equal volume of refold buffer (r
efolding buffer (3 M urea, 1 M Tris pH 7.3,
0.5 M L-arginine, 0.5 M NaCl, 0.1 M Na ₂ HPO ₄ , 1
mM reduced glutathione (GSH)) and incubated overnight at room temperature. The refolded 6-His endostatin was dialyzed extensively against PBS, pH 7.4 at room temperature. The resulting protein, 6-His endostatin, is soluble and analyzed by SDS-PAGE under non-reducing conditions (where 6-His
His endostatin was run as a single 20 kDa band) and was very pure.

B. Functional Activity of Endostatin In addition to the fact that the expressed protein is completely soluble, E. coli col
Other evidence that i-expressed 6-His endostatin is biologically active includes demonstrated binding to endothelial cells. Biotinylated 6-Hi
s endostatin was prepared and three different endothelial cell types (Be
ND3 mouse endothelial cells; ABAE, bovine aortic endothelial cells; and HUVEC, human umbilical cord endothelial cells). O-phenylenediamine (OP
Streptavidin-peroxidase in combination with D), and 490 nm
Detection was performed using the absorbance read in the section.

Direct binding studies have shown that (biotinylated 6-His) endostatin binds in vitro to three different types of endothelial cells in a supersaturated manner. It was also shown that expressed endostatin (without label) competes with biotinylated endostatin for binding to Bend3 endothelial cells.

C. Binding of Endostatin to 2C3 via SMPT and 2-IT 4-Succinimidyloxycarbonyl-α-methyl-α- (2-pyridyldithio) in N′N-dimethylformamide (DMF) ) -Toluene (SMPT
) Was added to 2C3 IgG at a molar ratio of 5: 1 (SMPT: 2C3) and incubated for 1 hour at room temperature (RT) in PBS with 5 mM EDTA (PBSE). Free SMPT was removed by G25 size exclusion chromatography performed with PBSE. Simultaneously, mouse 6-His endostatin was incubated with 2-iminothiolane (2-IT, Traut's reagent) at a molar ratio of 1: 5 (endostatin: 2-IT) for 1 hour at RT. Free 2
-IT was removed by size exclusion chromatography performed with PBSE.

[0851] SMPT modified 2C3 is mixed with 2-IT-modified 6-His endostatin,
Concentrated to 3-5 ml and incubated for 24 hours at RT with gentle shaking. Reactions were analyzed by SDS-PAGE. The unconjugated 2C3-SMPT is
Heparin affinity chromatography removes from the conjugate and removes 2C3
-Obtain endostatin.

D. Binding of Endostatin to 2C3 via SMCC and SATA N-Succinimidyl S-acetylthioacetate (SATA) was treated with 6-Hi
Incubation with s-endostatin at a molar ratio of 6: 1 (SATA: endostatin) for 30 minutes at room temperature. Free SATA was removed by G25 size exclusion chromatography performed with PBSE. SATA modified 6-H in PBSE
Is endostatin was concentrated to 4.0 ml and 0.4 ml deacetylated solution (0.1 M hydroxylamine) was added. The mixture was incubated at room temperature for 2 hours. At the same time, 2C3 IgG in PBSE was converted to succinimidyl
4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMC
Incubated with C) at a molar ratio of 1: 5 (2C3: SMCC). Free SMCC was removed by G25 size exclusion chromatography performed on PBSE.

The deacetylated SATA-modified endostatin was then incubated with SMCC-modified 2C3, concentrated under a stream of nitrogen to about 5 mg / ml total protein, and incubated overnight at RT with gentle agitation. The reaction was performed on SDS-PAG
Analyzed by E. Unconjugated 2C3-MCC is removed from the 2C3-endostatin conjugate by heparin affinity column chromatography in PBSE to yield 2C3-endostatin. Successful conjugation has also been achieved using 2-iminothiolane rather than SATA as the thiolating agent.

E. Fusion Protein of 2C3 and Endostatin 2C3-Endostatin fusion protein when DNA sequences for mouse and human endostatin, and 2C3 are available (and provided herein) Can be easily prepared. Endostatin expression and refolding, as described above, indicate that successful recombinant expression of this molecule is indeed possible.

[0853] Preparation of the 2C3-endostatin fusion protein can be in a form in which endostatin is present at the C-terminus of the heavy chain of 2C3 or linked to a 2C3 ScFv fragment. In these cases, recombination techniques directly alter the ligation and specifically envisage the use of selectively cleavable sequences to join the two functional parts. Plasmin cleavable or MMP cleavable sequences are presently preferred.

The efficacy of the selectively cleavable sequence and the adaptability of the recombinant technology are also demonstrated by the 2C3-
An endostatin fusion protein is provided, where endostatin is replaced with another part of the 2C3 construct. Endostatin can be selectively cleaved (
At this site, it is embedded in 2C3 until contacted with an enzyme that acts on the functional endostatin (released from the fusion protein).

(Example XI) (2C3-Ang-2 conjugate) (A. Expression of Ang-2) In order to construct a 2C3-Ang-2 conjugate, Ang-2 was expressed using a baculovirus expression system. If it can be produced in insect cells, it is preferably used in recombinant form.
A currently preferred protocol for Ang-2 expression and purification is Ang-2 cD
NA was cloned from mouse placental RNA by RT-PCR ^™ and
Cloning the g-2 cDNA into the pFastBac1 expression vector. Competent DH10Bac E. coli cells are transformed with the recombinant plasmid.

After antibiotic selection, E. coli containing recombinant Bacmid was selected. E. coli colonies are picked, expanded, and recombinant Bacmid DNA is purified. Insect cell SF9 was converted to Ce
Transfect with recombinant Bacmid DNA using 11fect reagent. Recombinant baculovirus is collected from the supernatant of transfected SF9 cells. The recombinant baculovirus is amplified and used to infect SF9 cells, and the infected SF9 cells express Ang-2. Ang-2 was purified from the supernatant of such infected SF9 cells by affinity purification.

B. Binding of Ang-2 to 2C3 Purified 2C3 was coupled to recombinant Ang-2 using the chemical linker SMPT, as described generally above. SMPT in N'N-dimethylformamide (DMF) is added to 2C3 IgG at a molar ratio of 5: 1 (SMPT: 2C3) and incubated for 1 hour at room temperature (RT) in PBS with 5 mM EDTA. . Free SMPT was removed by G25 size exclusion chromatography performed with PBSE. At the same time, the recombinant Ang-2 is incubated with 2-IT at room temperature. Free 2-IT was removed by G25 size exclusion chromatography performed with PBSE.

[0859] SMPT modified 2C3 is mixed with 2-IT modified recombinant Ang-2, concentrated, and incubated with gentle shaking for 24 hours. The reaction is analyzed by SDS-PAGE. Unconjugated 2C3-SMPT is removed from the conjugate by gel filtration chromatography to yield 2C3 endostatin.

Example XII 2C3-Tissue Factor Conjugate 2C3 was modified with SMTP as described in the above example. Free SMPT
SMPT was removed by G25 chromatography as outlined above except that the peak (2C3-SMPT) was collected under a stream of nitrogen. 600
μl of 2C3-SMPT was removed to quantify thiopyridine groups after addition of up to 50 mM dithiothreitol (DTT). An average of three MPT groups were introduced per IgG. Human truncated tissue factor (tTF) having a cysteine residue introduced at the N-terminus was reduced with 5 mM β2-ME. β2-ME,
Removed by G25 chromatography.

The reduced N-Cys-tTF was pooled with 2C3-SMPT and
. Incubated for 24 hours at RT at a molar ratio of 5: 1 (tTF: IgG).
The reaction was run on an Am with a 50,000 molecular weight cut off (MWCO) membrane.
It was concentrated to 1-2 ml using an icon. Unconjugated tTF and IgG are
The conjugate was removed using Superdex 200 size exclusion chromatography to yield 2C3-tTF.

Example XIII 2C3-CRM107 Conjugate 2C3 was modified with SMPT. Cytotoxic drug CRM107 (Jerry F
Dr. Ulton, Inland Laboratories, DeSoto, T
X) was modified with 2-IT as described in the above example. SMPT Modification 2C
3 was incubated with 2-IT modified CRM107 at a molar ratio of 1: 5 (IgG: CRM107) for 24 hours at RT with gentle shaking. Conjugated 2C3 is
The free reaction was removed using Superdex 200 size exclusion chromatography to yield 2C3-CRM107.

Example XIV 2C3 Prodrug Studies A. Cloning and Expression of β-Glucuronidase (GUS) The plasmid containing the E. coli GUS gene (pBacgus-1) was
agen, Inc. Obtained from. GU was transferred to the H6pQE60 expression vector.
The S gene, which encodes an N-terminal 6 × histidine tag, was used as a template for PCR to clone. E. coli harboring the plasmid col
iM15 cells were grown at a cell density of 560 nM to reach an optical density of 0.6. 0.1 mM isopropylthiogalactoside (IPTG) was added to induce 6 his GUS expression. After 4 hours, cells were collected by centrifugation.

[0866] E. The E. coli pellet is lysed with a cell lysis buffer (B-PER Bacterial).
Protein Extraction Reagent (Pierce, R
ockford, IL)). The solution was loaded onto a Ni-NTA column, the column wash buffer _{(6M GuHCl, 100mM NaH 2 PO} 4,
(10 mM Tris, 500 mM NaCl, pH 7.3) and the bound 6-His GUS was eluted from the column with 0.2 M imidazole in the same buffer.

[0866] 6-His GUS is pure based on SDS-PAGE and 6-His GUS
GUS migrated as a single band of the 75 kDa band. In a gel filtration column, 6-His GUS runs as a tetramer of approximately 300 kDa. 6-His GUS is enzymatically active as judged by its ability to cleave the substrate p-nitrophenyl-β-D-glucuronide (PNPG).

(B. Binding of 2C3 to β-glucuronidase (GUS)) N-succinimidyl S-acetylthioacetate (SATA) was mixed with GUS at a molar ratio of 6: 1 (SATA: GUS) at room temperature for 30 minutes. Incubated for minutes. Free SATA was removed by G25 size exclusion chromatography performed with PBSE. SATA modified GUS in PBSE was concentrated to 4.0 ml and 0.4 ml deacetylated solution (0.1 M hydroxylamine) was added.
The mixture was incubated at RT for 2 hours. At the same time, 2C3 I in PBSE
gG was converted to succinimidyl 4- (N-maleimidomethyl) cyclohexane-1
-Incubation with carboxylate (SMCC) at a molar ratio of 1: 5 (2C3: SMCC). Free SMCC was removed by G25 size exclusion chromatography performed on PBSE.

[0868] The deacetylated SATA modified GUS was then incubated with SMCC modified 2C3 overnight at RT with gentle agitation. Free GUS was removed by ion exchange chromatography on Q-Sepharose along with free 2C3, and the 2C3-GUS conjugate was eluted with 0.5M NaCl in PBS.
The resulting solution was separated by Superdex 200 size exclusion chromatography to give 2C3-GUS with 90% purity.

(C. Biological Activity of 2C3-GUS Conjugate) The biological activity of each component of the 2C3-GUS conjugate was confirmed. 2C3-GUS specifically binds to VEGF-coated wells in a well-controlled ELISA as detected by secondary HRP-labeled anti-mouse IgG and OPD. Half-maximal binding was observed at 0.1 nM. Thus, the 2C3 binding moiety functions correctly.
The GUS moiety also retained enzyme activity.

[0870] 2C3-GUS was radioiodine labeled with ¹²⁵ I to a specific activity of 5 × 10 ⁶ cpm / μg. After intravenous injection into mice, radioiodine labeled 2C3-GUS was
Blood was purged at t1 / 2α for 6 hours and t1 / 2β for 25 hours.

D. GUS-cleaved prodrugs β-glucuronide prodrugs (eg, doxorubicin-β-glucuronide and calcimicin-β-glucuronide) are described in US Pat. No. 5,561,119 (herein incorporated by reference). Prepared essentially as described in (Incorporated). Such prodrugs are designed to release cytotoxic components (eg, doxorubicin or calcimicin) only when digested by a glucoside enzyme (eg, GUS). By attaching GUS to 2C3, GUS becomes
It specifically targets tumor vasculature and stroma, providing for specific cleavage of prodrugs and release of certain cytotoxic components within the tumor site.

E. Biological Activity of 2C3-GUS Conjugates 2C3-GUS was administered to SCID mice bearing human NCI-H358 NSCLC tumors at subcutaneous sites. v. After injection, it is specifically localized around the tumor vasculature and tumor stroma. The presence of 2C3-GUS was detected immunohistochemically in frozen sections of tumors using HRP-labeled anti-mouse IgG or HRP-labeled anti-GUS. Maximum localization was observed 24-48 hours after injection of 2C3-GUS. Normal tissues were not stained. 2C3-G around tumor vasculature and tumor stroma
The specific localization of the US activates systemically administered prodrugs (eg, doxorubi single clonide or calcimicin-glucuronide) only within the tumor.

[0873] Hybridoma cell lines as described herein were prepared under the provisions of the Budapest Treaty by the American Type Culture Collection (ATCC), Mana.
sassas, VA, USA; and accession number ATCC number PTA 159
5 was assigned. The invention described and claimed herein is not limited in scope by the deposited PTA 1595 cell line. Because the deposited embodiments illustrate one aspect of the present invention, equivalent hybridoma cell lines that functionally produce equivalent monoclonal antibodies are included within the scope of the present invention. Indeed, all compositions and methods described and claimed herein can be made and completed without undue experimentation in light of the present disclosure.

The compositions and methods of the present invention are described in the preferred embodiments section, but the compositions and methods described herein and the steps or series of steps of the methods of the present invention are It will be apparent to those skilled in the art that modifications may be made without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain reagents, both chemical and physiological, may be substituted for the agents described herein, but the same or similar results will be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

References The following references are hereby incorporated by reference in their entirety to the extent that they provide exemplary procedures or other details that supplement the description herein.

[0876]

[Table 11] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings, in conjunction with the detailed description of the specific embodiments set forth herein.

[Sequence list]

[Brief description of the drawings]

FIG. 1. 2C3 inhibits VEGF-mediated proliferation of ABAE cells. ABAE cells are grown in the presence of various indicated antibodies and 0.5 nM VEGF. Growth after 4 days was determined colorimetrically by enzymatic conversion of MTS (Owen's reagent) to yellow formazan. Results are shown as percentage formazan production in control wells grown with VEGF alone. Background is VEGF
Or determined by growing cells without antibody and subtracted from control wells and sample wells. The results show the arithmetic mean of three measurements, the standard deviation of which was always less than 20% of the mean. Growth curves for anti-VEGF IgG antibodies (mAb 4.6.1 as a positive control and irrelevant specific IgG as a negative control (control IgG)) were shown.

FIG. 2C3 shows V vs. VEGFR2 but not VEGFR1 in ELISA
EGF binding was blocked. Wells are coated with the extracellular domain of VEGFR1 (Flt-1 / Fc) or VEGFR2 (sFlk-1) and then the indicated IgG presence of either 1 nM alone VEGF or 100 nM or 1000 nM. Incubated below with VEGF. The plate was then coated with 1 μg / ml rabbit anti-VEGF (A-20, Santa Cruz).
Biotechnology, Inc. ) And generated using a peroxidase-conjugated goat anti-rabbit antibody. The assay was performed three times. In the absence of antibody, the average percentage of binding was shown with standard deviation. The asterisk indicates a value that is statistically significantly different from the value in the absence of the antibody (p <0.002) by the Student's T test.

3A and 3B inhibit the in vivo growth of human tumor xenografts. FIG. 3A: 1 × 10 ⁷ NCI-H358 NSCLC cells were injected subcutaneously in nu / nu mice on day 0.
Injected. FIG. 3B: 5 × 10⁶ A673 rhabdomyosarcoma cells were nu at day 0
/ Nu mice were injected subcutaneously. The mice are then given once daily and twice weekly.
The injected IgG was injected intraperitoneally. 2C3 has a dose of 100, 10, or 1
Control IgG given in μg / injection, while irrelevant specificity (FIG. 3A)
And 3E7 (FIG. 3B) were also given at 100 μg / injection. Tumor, one week
Measured 2-3 times per interval. Means and standard errors are shown in FIG. 3A for this study.
Data for the last day of this study (day 26).
The data is shown in FIG. 3B.

FIG. 3B. 2C3 inhibits in vivo growth of human tumor xenografts. FIG. 3A: 1 × 10 ⁷ NCI-H358 NSCLC cells were injected subcutaneously in nu / nu mice on day 0.
Injected. FIG. 3B: 5 × 10⁶ A673 rhabdomyosarcoma cells were nu at day 0
/ Nu mice were injected subcutaneously. The mice are then given once daily and twice weekly.
The injected IgG was injected intraperitoneally. 2C3 has a dose of 100, 10, or 1
Control IgG given in μg / injection, while irrelevant specificity (FIG. 3A)
And 3E7 (FIG. 3B) were also given at 100 μg / injection. Tumor, one week
Measured 2-3 times per interval. Means and standard errors are shown in FIG. 3A for this study.
Data for the last day of this study (day 26).
The data is shown in FIG. 3B.

FIG. 4. 2C3 treatment reduces the size of established human NCI-H358 NSCLC tumor xenografts. Mice held subcutaneous NCI-H358 tumors (size approximately 300~450mm ³⁾ is at the indicated time points, the 50μg or 100 [mu] g, 2
Treated intraperitoneally with C3 (n = 14), mAb 4.6.1 (n = 5), 3E7 (n = 12) or control IgG (n = 9). Average tumor volume is shown with SEM over 116 days.

FIG. 5: Comparison of 2C3 and 2E3 treatment of established human tumor xenografts. FIG. 5A: Mouse subcutaneous NCI-H358 tumor (approximately 450 mm ^{3 in} size) shows 2C3
(N = 6) or 3E7 (n = 4). Treatment (T) is 100 μg IgG given intraperitoneally, except for the first treatment consisting of 500 μg IgG given intravenously. The mean tumor volume is shown along with the SEM. At the end of the study (day 116), mice are sacrificed, and tumors are analyzed and weighed. The average tumor weight for the 2C3 treated group was 0.054
g, but the 3E7 treatment group had an average tumor weight of 0.545 g. FIG. 5B shows a mouse-bearing subcutaneous HT1080 tumor (approximately 200-250 mm ^{3 in} size).
Is 100 μg of 2C3 (n = 9), 3E7 (n = 11), control IgG
(N = 11), or with saline (n = 11) intravenously. Mice were treated every other day as indicated (T). Non-2C3-treated mice were sacrificed on day 26 due to more than 50% of each group having large, ulcerated tumors. The mean tumor volume is shown along with the SE.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 11, 2001 (2001.1.11)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0166

[Correction method] Change

[Contents of correction]

Tumor targeting and treatment with coag ligands are described in the following patents and patent applications, each of which is incorporated herein by reference to yet further augment the present teachings, particularly with respect to coag ligands and coagulation factors: No. 07 / 846,349; 08 / 205,330 (U.S. Pat.
No. 866); 08 / 350,212 (U.S. Pat. No. 5,965,132).
; Nos 08 / 273,567; the No. 08 / 482,369 (US Patent No. 6, 093,399 Nos; Nos 08 / 485,482; the No. 08 / 487,427 (US Patent No. 08 / 479,733 (U.S. Patent No. 5,877,289); and 08 / 472,631; 08/4.
Nos. 79,727 and 08 / 481,904 (U.S. Pat.
No. 55).

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0178

[Correction method] Change

[Contents of correction]

The following patents and patent applications are each incorporated herein by reference for the purpose of further supplementing the present teachings on coag ligand preparation, purification and use, including bispecific antibody coag ligands. U.S. Application No. 07/846,
349; 08 / 205,330 (U.S. Pat. No. 5,855,866); 08/3.
50,212 (US Pat. No. 5,965,132); 08 / 273,567; 0
8 / 482,369 (U.S. Patent No. 6,093,399; 08 / 485,482).
08 / 487,427 (U.S. Patent No. 6,004,555); 08/479,
733 (U.S. Pat. No. 5,877,289); 08 / 472,631; and 0
8 / 479,727 and 08 / 481,904 (US Pat. No. 6,036,955).
No. 5).

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0423

[Correction method] Change

[Contents of correction]

The following patents and patent applications disclose the present teachings regarding the preparation and use of functional antigen-binding regions of antibodies, including scFv, Fv, Fab ′, Fab and F (ab ′) ₂ fragments of anti-VEGF antibodies. No. 5,855,866; US Pat. No. 5,965,132; US Pat. No. 6,051,230 for the purpose of still further supplementing US Pat. No. 6,004,555; and No. 5,877,289; and US patent application Ser. No. 08 / 482,3.
No. 69 (Payment fee was paid on October 20, 1998). WO98 /
45331 also describes the variable, hypervariable, and complementarity determining regions (CD
R ) is incorporated herein by reference for purposes including including and further describing the preparation of R ) .

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0427

[Correction method] Change

[Contents of correction]

A modest binding modification for use with the present invention involves incorporating a salvage receptor binding epitope into the antibody fragment. Techniques for accomplishing this include mutation of the appropriate region of the antibody fragment, or incorporation of the epitope as a peptide tag attached to the antibody fragment. WO9
No. 6/32478 is specifically incorporated herein by reference for the purpose of further describing such techniques. Salvage receptor binding epitope is typically a one or two loops (lo o p) regions of three or more amino acids from the Fc domain are transferred to a similar position in the antibody fragment. WO98 /
The 45331 salvage receptor binding epitope is hereby incorporated by reference for use with the present invention.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0703

[Correction method] Change

[Contents of correction]

The following patents and patent applications are specifically incorporated herein by reference for the purpose of still further complementing the teachings of the present invention regarding the preparation and use of agents that target tumor stroma. : U.S. Patent No. 5,877,289 and U.S. Patent Application No. 08 / 482,369 (U.S. Patent Nos. 6,093,399; 08/485).
No. 08 / 487,427 (US Pat. No. 6,004,555).
No. 08 / 479,733 (U.S. Pat. No. 5,877,289);
Nos./472,631 and 08 / 479,727 and 08/481.
, 904 (U.S. Patent No. 6,036,955).

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] May 9, 2001 (2001.5.9)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0007

[Correction method] Change

[Contents of correction]

[0007] Another effective version of the vascular targeting approach is to target clotting factors to markers expressed or adsorbed in cellular blood vessels (Huang et al., 1).
997; U.S. Patent 5,877,289, the No. 6,004,555, and the No. 6,093. Delivering coagulants, rather than toxins, to tumor blood vessels,
It has the added advantage of reduced immunogenicity and a lower risk of toxin side effects. As described in US Pat. No. 5,877,289, preferred coagulation factors for use in such tumor-specific "coagulants" include truncated forms of human coagulation-inducing protein, tissue factor (TF), blood coagulation Is the main initiator.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 47/48 A61K 47/48 4C085 A61P 9/10 A61P 9/10 4H045 27/02 27/02 35/00 35/00 43/00 105 43/00 105 111 111 123 123 C07K 16/30 C07K 16/30 C12N 5/02 C12N 5/02 5/10 ZNA C12P 21/08 15/02 C12N 5/00 ZNAB C12P 21 / 08 15/00 C (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, S, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventors Brecken, Rolf A. United States Washington 98125, Seattle, 25TH Avenue NE 14304 F-term (Reference) 4B024 AA01 AA11 BA45 BA61 CA04 CA07 GA01 GA04 GA11 GA18 HA08 HA11 4B064 AG26 AG27 DA01 DA14 4B065 AA90X AA90Y AA93Y AA95X AB02 AB04 CC24 A FF68 4C084 AA17 NA13 NA15 ZA331 ZB212 ZB262 4C085 AA14 AA21 AA25 AA26 AA27 CC02 4H045 AA10 AA11 AA20 AA30 BA09 BA42 BA54 CA40 DA75 DA76 DA86 EA20 EA50 EA51

Claims (85)

[Claims] 1. A composition comprising a biologically effective amount of an anti-VEGF antibody, or an antigen-binding fragment thereof, wherein the antibody comprises the monoclonal antibody ATCC PT.
A 1595 binds to substantially the same epitope as VEGF receptor VE
VEG without significant inhibition of VEGF binding to GFR1 (Flt-1)
A composition which significantly inhibits the binding of VEGF to the F receptor VEGFR2 (KDR / Flk-1). 2. The composition of claim 1, wherein said antibody is a human, humanized, partially human, chimeric or recombinant antibody or antigen-binding fragment thereof. 3. The antibody of claim 1 wherein said antibody is a monoclonal antibody produced by a hybridoma deposited as ATCC PTA 1595.
A composition according to claim 1. 4. The composition according to claim 1, wherein said antibody is operably linked to at least a first biological agent. 5. The method of claim 4, wherein the antibody is operably linked to at least a first therapeutic agent or an agent that cleaves a substantially inactive prodrug to release a substantially active drug. A composition as described. 6. The method of claim 1, wherein the antibody comprises at least a first chemotherapeutic agent, a radiotherapeutic agent, an anti-angiogenic agent, an apoptosis-inducing agent, an anti-tubulin agent, a coagulant, a cytotoxic agent, a cytostatic or an anti-proliferative agent. 6. The composition of claim 5, wherein the composition is operably linked to a cellular agent. 7. A composition according to claim 1, for use in therapy. 8. A composition according to any of claims 1 to 7 for use in inhibiting angiogenesis in an animal having an ocular neovascular disease, macular degeneration or angiogenic tumor. 9. Use of a composition according to claims 1 to 8 in therapy. 10. The method according to any one of claims 1 to 8, in the manufacture of a medicament for the treatment of ocular neovascular diseases, macular degeneration or cancer by inhibiting angiogenesis.
Use of a composition as described in paragraph. 11. The method according to claim 11, wherein the antibody is a monoclonal antibody ATCC PTA 15
A composition according to claim 1, wherein the composition is 95. 12. The composition of claim 1, wherein the antibody is operably linked to at least a first biological agent. 13. The antibody is operably linked to at least a first agent that cleaves a substantially inactive prodrug to release a substantially active drug.
The composition according to claim 12. 14. The composition of claim 13, wherein said antibody is operably linked to alkaline phosphatase which cleaves a substantially inactive phosphate prodrug to release a substantially active drug. 15. The composition of claim 12, wherein said antibody is operably linked to at least a first therapeutic or diagnostic agent. 16. The composition of claim 15, wherein said antibody is operably linked to at least a first therapeutic agent. 17. The composition of claim 16, wherein said antibody is operably linked to at least a first therapeutic agent and a second therapeutic agent. 18. The method of claim 18, wherein the antibody comprises at least a first chemotherapeutic agent, a radiotherapeutic agent,
17. An operatively linked anti-angiogenic agent, apoptosis inducer, steroid, antimetabolite, anthracycline, vinca alkaloid, anti-tubulin drug, antibiotic, cytokine, alkylating agent or coagulant. 18. The composition according to 17. 19. The antibody is operably linked to a cytotoxic, cytostatic or anticellular agent capable of killing endothelial cells or inhibiting endothelial cell proliferation or cell division. The composition of claim 18. 20. The composition of claim 19, wherein said antibody is operably linked to a plant, fungal or bacterial toxin. 21. The antibody according to claim 20, wherein the antibody is an A-chain toxin, a ribosome-inactivating protein,
21. The composition of claim 20, wherein the composition is operably linked to sarsine, gelonin, aspergillin, restrictocin, ribonuclease, epipodophyllotoxin, diphtheria toxin or Pseudomonas exotoxin. 22. The composition of claim 21, wherein said antibody is operably linked to a ricin A chain or a deglycosylated ricin A chain. 23. The composition of claim 18, wherein said antibody is operably linked to an anti-angiogenic agent. 24. The antibody, wherein the antibody is operably linked to angiopoietin.
A composition according to claim 23. 25. The composition of claim 24, wherein said antibody is operably linked to angiopoietin-2 and angiopoietin-1. 26. The composition of claim 23, wherein said antibody is operably linked to angiostatin, vasculostatin, canstatin, or maspin. 27. The composition of claim 23, wherein said antibody is operably linked to endostatin. 28. The antibody, wherein the antibody is operably linked to an anti-tubulin agent.
A composition according to claim 18. 29. The method according to claim 29, wherein the antibody is colchicine, taxol, vinblastine,
29. The composition of claim 28, wherein the composition is operably linked to an anti-tubulin drug selected from the group consisting of vincristine, vindesine and combretastatin. 30. The antibody of claim 18, wherein the antibody is operably linked to a coagulant.
A composition according to claim 1. 31. The composition of claim 30, wherein said antibody comprises:
Factors II / IIa, VII / VIIa, IX / IXa, X / X
Factor a, vitamin K-dependent coagulation factor lacking Gla modification, Russell's viper snake venom factor X activator, thromboxane A ₂ , thromboxane A ₂ synthase and α2
-A composition operably linked to a coagulant selected from the group consisting of antiplasmin. 32. The antibody, wherein the antibody lacks the ability to activate tissue factor, human tissue factor, factor VII, mutant tissue, truncated tissue factor or dimeric, trimer, or polymeric tissue factor. 31. The composition of claim 30, wherein the composition is operably linked to a tissue factor derivative. 33. The composition of claim 32, wherein said antibody is operably linked to truncated tissue factor. 34. The composition of claim 15, wherein said antibody is operably linked to a diagnostic, imaging or detectable agent. 35. The composition of claim 34, wherein said antibody is operably linked to an X-ray detectable compound, radioactive ion or nuclear magnetic spin resonance isotope. 36. The composition of claim 34, wherein said antibody is operably linked to an enzyme that produces a colored product upon contact with biotin, avidin, or a chromogenic substance. 37. The composition according to any one of claims 12 to 33, wherein the antibody is operably linked to the biological agent as a fusion protein, wherein the fusion protein comprises: Prepared by expressing a recombinant vector,
A composition wherein the recombinant vector comprises, in the same reading frame, a DNA segment encoding the antibody operably linked to a DNA segment encoding the biological agent. 38. The antibody of claim 1, wherein the antibody is operably linked to the biological agent via a biologically dissociable bond or a selectively cleavable linker.
34. The composition according to any one of items 2 to 33. 39. The composition of claim 38, wherein said antibody is operably linked to said biological agent via a peptide linker, said peptide linker comprising urokinase, prourokinase, plasmin, Plasminogen, TG
A composition comprising a cleavage site for Fβ, staphylokinase, thrombin, factor IXa, factor Xa, metalloproteinase, stromal collagenase, gelatinase or stromelysin. 40. The composition of any one of claims 12-36, wherein said antibody is directly linked to said biological agent. 41. The composition of any one of claims 15-36, wherein said antibody is operably linked to a second antibody that binds to said therapeutic or diagnostic agent or an antigen binding region thereof. . 42. The composition according to any one of claims 1 to 8 or 11 to 41, wherein said composition is a pharmaceutically acceptable composition. 43. The composition of claim 42, wherein said pharmaceutically acceptable composition is formulated for parenteral administration. 44. The composition of claim 1, wherein said composition further comprises a second therapeutic agent.
Or the composition according to any one of 11 to 43. 45. The composition of claim 44, wherein said second therapeutic agent is a second anti-cancer agent. 46. The second anticancer agent is a chemotherapeutic agent, a radiotherapeutic agent, an antiangiogenic agent, an apoptosis inducing agent, an antitubulin drug or a tumor-targeting chemotherapeutic agent, a radiotherapeutic agent, an antipulse. 46. The composition of claim 45, which is a tube forming agent, an apoptosis inducer or an anti-tubulin agent. 47. The composition of claim 46, wherein said second anticancer agent is angiopoietin or a tumor targeting angiopoietin. 48. The composition of claim 47, wherein said second anti-cancer agent is angiopoietin-1 targeting a tumor. 49. The composition of claim 46, wherein said second anti-cancer agent is endostatin or a tumor-targeting endostatin. 50. The composition of claim 46, wherein said second anti-cancer agent is an anti-tubulin drug or an anti-tubulin drug targeting a tumor. 51. The composition of claim 46, wherein said second anticancer agent comprises a therapeutic agent operably linked to a second antibody or antigen-binding fragment thereof. A composition wherein the construct binds to a surface expressed component, a surface accessible component or a surface localization component of a tumor cell, tumor vasculature or tumor stroma. 52. The use according to claims 1 to 8 or 11 to 5 for use in therapy.
A composition according to any one of the preceding claims. 53. A method according to claim 1, for use in diagnosis.
The composition according to any one of items 4 to 36. 54. The composition according to any one of claims 1 to 8, 11 or 23 to 29 for use in inhibiting angiogenesis by administration to an animal. 55. The composition of claim 54, wherein the animal has ocular neovascular disease or macular degeneration for use in administering to an animal to inhibit angiogenesis. ,Composition. 56. The method according to any one of claims 12 to 41 for administration to an animal having a vascularized tumor to be used in delivering a biological agent to said vascularized tumor. Composition. 57. The use of claims 1-8 or 1 for use in treating cancer.
57. The composition according to any one of 1-56. 58. Use of a composition according to claims 1 to 8 or claims 11 to 57 in therapy. 59. Use of a composition according to any one of claims 1 to 8, 11 or 23 to 29 in the manufacture of a medicament for treating a disease by inhibiting angiogenesis. . 60. The use of claim 59, wherein said medicament is intended to treat ocular neovascular disease or macular degeneration by inhibiting angiogenesis. 61. Use of a composition according to any one of claims 12 to 41 in the manufacture of a medicament for treating cancer by delivering a biological agent to a vascularized tumor. 62. Use of a composition according to any one of claims 1 to 8 or 11 to 57 in the manufacture of a medicament for treating cancer. 63. A composition for use in inhibiting angiogenesis without substantially inhibiting macrophages, osteoclasts or cartilage resorbing cells, wherein the composition comprises a biologically active agent. A composition comprising an amount of an anti-VEGF antibody, or an antigen-binding fragment thereof, wherein the antibody binds substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). 64. VE to VEGF receptor VEGFR1 (Flt-1)
Without significantly inhibiting the binding of GF, the VEGF receptor VEGFR2 (KD
Use of a composition in the manufacture of a medicament for treating cancer by inhibiting the binding of VEGF to R / Flk-1), wherein the composition comprises a biologically effective amount of an anti-V
Use comprising an EGF antibody, or an antigen-binding fragment thereof, wherein said antibody binds to substantially the same epitope as monoclonal antibody 2C3 (ATCC PTA 1595). 65. A kit comprising at least the first composition according to any one of claims 1 to 8 or 11 to 57. 66. A kit comprising at least a first composition according to claim 13 or at least a second composition comprising a substantially inactive prodrug, wherein the prodrug is the first A kit that is cleaved by the biological agent linked to the antibody in one composition to release a substantially active drug. 67. A kit comprising at least a first composition according to claim 14 and at least a second composition comprising combretastatin phosphate. 68. A hybridoma producing an anti-VEGF monoclonal antibody, which binds to substantially the same epitope as monoclonal antibody ATCC PTA 1595, and binds to the VEGF receptor VEGFR1 (Flt-1
) Without significantly inhibiting the binding of VEGF to the VEGF receptor VEGF
A hybridoma that significantly inhibits VEGF binding to R2 (KDR / Flk-1). 69. The monoclonal antibody ATCC PTA 1595. 70. A method for preparing an anti-VEGF antibody that binds substantially the same epitope as monoclonal antibody ATCC PTA 1595, wherein the non-human animal comprises at least a first immunogenic VEGF component. Immunizing with the monoclonal antibody ATCC P from the immunized animal.
Selecting an antibody that substantially cross-reacts with TA 1595. 71. The method of claim 70, wherein said non-human animal is a transgenic mouse comprising a human antibody library. 72. The method of claim 70 or 71, comprising the steps of obtaining a nucleic acid encoding the anti-VEGF antibody, and expressing the nucleic acid to obtain a recombinant anti-VEGF antibody. 73. The method according to any one of claims 70 to 72, wherein
(A) administering to a non-human animal an immunologically effective amount of a composition comprising at least a first immunogenic VEGF component; (b) expressing RNA isolated from the spleen of the immunized animal (C) selecting from the phagemid library a clone that expresses an anti-VEGF antibody that substantially cross-reacts with monoclonal antibody 2C3 (ATCC PTA 1595); and And b) expressing a nucleic acid encoding an anti-VEGF antibody derived from the selected clone to produce a recombinant anti-VEGF antibody. 74. A method for detecting VEGF, comprising: forming a composition suspected of containing VEGF under conditions effective to permit formation of a VEGF / antibody complex. 58. A method comprising contacting with the composition of any one of claims 11 to 57, and detecting the complex thus formed. 75. A method for inhibiting VEGF binding to VEGF receptor VEGFR2 without significantly inhibiting VEGF binding to VEGF receptor VEGFR1, comprising VEGFR2 (KDR / Flk-1) and VEGFR
58. A method comprising contacting a homogeneous or heterogeneous population of cells expressing 1 (Flt-1) with a biologically effective amount of a composition according to claims 1-8 or 11-57. 76. A method for specifically inhibiting VEGF-induced endothelial cell proliferation, wherein the composition according to any one of claims 1-8 or 11-57 comprises a biologically effective amount of the endothelial cell population. Contacting with a method. 77. A method of inhibiting VEGF-induced endothelial cell proliferation without significantly inhibiting VEGF-induced macrophage function, osteoclast function, or cartilage resorbing cell function, the method comprising the steps of: 58. A method comprising contacting a tissue comprising at least one of the resorbing cells with a biologically effective amount of a composition according to any one of claims 1-8 or 11-57. 78. A method of inhibiting angiogenesis, comprising contacting a population of blood vessels with an anti-angiogenic composition, wherein the angiogenic composition has a biologically effective amount. 58. A method comprising the composition of any one of claims 1-8 or 11-57. 79. A method for treating an angiogenic disease, comprising the step of administering at least a first pharmaceutical composition to an animal having an angiogenic disease, 58. A method wherein the composition comprises a therapeutically effective amount of a composition according to any one of claims 1-8 or 11-57. 80. The animal has ocular neovascular disease or macular degeneration,
80. The method of claim 79. 81. A method for delivering a diagnostic or therapeutic agent to a vascularized tumor, comprising administering to the animal having the vascularized tumor an effective amount of a biologically effective amount.
A method comprising administering a composition according to any one of the preceding claims. 82. A method for treating cancer, wherein at least the first pharmaceutical composition is at risk of having or developing metastasis from a vascularized solid tumor, metastatic tumor or primary tumor. Administering to an animal, wherein said at least a first pharmaceutical composition comprises a therapeutically effective amount of a composition according to any one of claims 1-8 or 11-57. ,Method. 83. The method of claim 81 or 82, further comprising subjecting said animal to radiation therapy. 84. A method for treating cancer, comprising: (a) replacing the first composition of claim 13 or 14 with a vascularized solid tumor;
Administering to an animal having a metastatic tumor or a metastasis from a primary tumor, thereby localizing the antibody of the composition to the tumor vasculature or stroma; and (b) subsequently administering to the animal Administering a second composition comprising a substantially inactive prodrug cleaved by the biological agent linked to the antibody in the first composition, thereby providing a substantially active drug. Specifically releasing into the tumor vasculature or stroma. 85. The method of any one of claims 79 to 84, wherein said animal is a human patient.